Maturase K forms a plastidial splicing complex with a neofunctionalized branching enzyme

Domain: biology
Published: 2026-03-23
Editorial outcome: minor_revision

Abstract
Chloroplast group IIA introns originate from bacterial ribozymes. Their splicing requires the
splicing factor Maturase K (MatK). MatK, however, has been difficult to functionally analyse, as
it appears essential for plant viability and is encoded in the chloroplast genome. Here we
identify a heteromultimeric complex comprising MatK and three other essential, plastid-targeted
proteins using co-immunoprecipitation experiments in Arabidopsis and tobacco. Among the
MatK interactors is a conserved homologue of starch-branching enzymes (BEs), which we
name MATURASE K INTERACTING PROTEIN1 (MKIP1). We demonstrate that MKIP1 proteins
have lost BE activity and acquired a 150-amino acid insertion that enables direct interaction with
MatK’s N-terminus. Immunoprecipitation of Arabidopsis MKIP1 co-precipitates all known MatK
intron targets. Inducing MKIP1 silencing in Arabidopsis causes newly emerging leaves to be
pale, in which the splicing of MatK intron targets is strongly reduced. Our data suggest that
MKIP1 functionally diverged from canonical BEs to facilitate splicing in conjunction with MatK. In
turn, the former reverse-transcriptase domain in the N-terminal region of MatK likely has
acquired the capacity to interact with other proteins. Potentially, complex formation allowed
MatK to diversify its RNA interactions, helping its transition towards a general splicing factor.

Introduction
Chloroplast genomes (plastomes) of land plants contain ~20 introns. As they interrupt the
sequences of genes required for photosynthesis, protein homeostasis, and for plastidial gene
expression itself, intron splicing is an indispensable stage of plastidial RNA processing and its
failure usually results in plant inviability1,2. Most plastidial introns belong to group II introns
derived from mobile bacterial elements, which are also probable predecessors of nuclear mRNA
introns3. Much of our understanding of the mechanism of plastidial intron splicing derives from
analogy to the splicing of these bacterial ancestors. All group II introns share an RNA structure
consisting of six double-helical domains (denoted DI-DVI)4. In bacteria, these were shown to
fold into a complex tertiary structure that allows the RNA to splice via two consecutive splicing
steps, resulting in a branched intron lariat and ligated exons5. However, although splicing is
catalyzed by the ribozyme intron RNA itself, efficient splicing of bacterial introns usually requires
a maturase protein6. These are typically encoded within intron domain IV and have an N-
terminal reverse transcriptase (RT) domain, followed by a maturase (X) domain, among others7.

During splicing, the RT domain of the bacterial maturase stably docks the protein onto the intron
in a sequence-specific manner, allowing the X domain to make flexible contacts with the intron
core7–9. The X domain then facilitates critical RNA conformations during splicing by stabilizing
the intron’s catalytic core and the position of the bulged adenosine in domain VI, the nucleophile
of the first splicing step10,11.

Bacterial group II introns each encode their own, highly specific maturase. Contrarily, in land
plants chloroplasts, only the trnK intron encodes a maturase-like protein, Maturase K (MatK),
while the other group II introns do not. Tobacco MatK was observed to bind to six or seven
introns, i.e. to all group IIA introns except the divergent introns in clpP and (possibly) trnV12–15.
MatK likely helps the splicing of its binding targets, since splicing is reduced upon MatK
reduction by genetic or chemically-induced disruption of plastid translation, and since matK
gene loss coincides with intron loss in several parasitic plants16. These observations imply that
MatK has evolved into a general splicing factor, but direct functional analyses of MatK have
been hindered by the lack of (hypomorphic) mutants of this likely essential gene12.

Consistent with a potential functional diversification, MatK has structurally diverged from
bacterial maturases. It retained its X domain, indicating that it can still interact with the intron
core facilitating intron splicing17. However, its RT domain appears degenerated, since the N-
terminal RT motifs of bacterial maturases are no longer recognizable17. This includes most of
RT0 region which enables bacterial maturases to form strong and sequence-specific
interactions with intron domain IV8,9,18. The potential loss of specific RNA binding may have
helped MatK to act on multiple introns but likely comes at the cost of a lower RNA affinity and
may negatively affect its orientation on the intron7. In addition, the plastidial introns have lost
RNA-RNA interaction motifs involved in the folding of bacterial introns19. These deviations may
explain why plastidial group IIA intron splicing requires an array of nuclear-encoded splicing
factors, such as CRS1 (Chloroplast RNA Splicing 1), RNC1 and WTF1 (“what’s this factor?”)20–
22. These possibly assist in the initial folding and stabilization of the precursor1,2, but have not
been reported to associate directly with MatK.

Here we report a protein that physically interacts with MatK to help plastidial intron splicing in
Arabidopsis. This protein was previously named BRANCHING ENZYME1 (AtBE1)
(AT3G20440) based on its homology to starch-branching enzymes23 (BEs), which introduce α-
1,6-linkages into the polymers comprising starch, a plastidial storage carbohydrate24. However,
in contrast to the other two Arabidopsis BEs (AtBE2 and AtBE3), AtBE1 has no apparent BE
activity23,25. In addition, Arabidopsis be1 mutants are embryo defective and inviable26,27, while
be2 and be3 single mutants are phenotypically normal23. The be2/be3 double mutant lacks
starch and grows poorly but is viable23, as are, to our knowledge, all other Arabidopsis mutants
with primary defects in starch metabolism described to date. We show that AtBE1 is a
conserved, neofunctionalized BE that has acquired a new role in plastid intron splicing by
forming a splicing complex with AtMatK. To recognize its function and avoid confusion with
canonical BEs, we renamed it to MATURASE K INTERACTING PROTEIN1, or MKIP1.

Results
AtMKIP1 is required for plant viability
We reconfirmed that AtMKIP1 is essential for embryo development26,27 using two lines (mkip1-1
and mkip1-2) carrying T-DNA insertions in introns 15 and 16 of the AtMKIP1 (AT3G20440)
gene, respectively (Fig. 1a, Supplementary Fig. 1). No mkip1-/- mutant plants could be
identified from the progeny of mother plants heterozygous for either allele (192 and 100 plants
analyzed for mkip1-1 and mkip1-2, respectively). In addition, siliques from heterozygous plants
contained ~25% white seeds with embryos arrested at the heart stage (Fig. 1a, Supplementary
Fig. 1). These phenotypes could be complemented by UBQ10-driven, constitutive expression of
the coding sequence (CDS) of AtMKIP1 fused to a C-terminal mCitrine or eYFP (named YFP
hereafter) tag in heterozygous mkip1-1 or mkip1-2 plants. Complemented mkip1-1 -/- and mkip1-
2-/- plants appeared wild-type (WT) like, although they grew slightly slower (Fig. 1,
Supplementary Figs. 1, 2c). Confocal microscopy of 35S-driven, stable expression of
AtMKIP1-YFP in WT Arabidopsis confirmed that AtMKIP1 exclusively localizes to the
chloroplasts of epidermal cells and shows a fairly homogeneous distribution within the
chloroplast stroma26 (Fig. 1b, Supplementary Fig. 3).

In an attempt to bypass the embryonic arrest of mkip1-/- mutants, we transformed heterozygous
mkip1-1+/- and mkip1-2+/- plants with a construct expressing AtMKIP1-mCitrine under the control
of the embryo-specific ABI3 promoter28. Siliques from mkip1+/- plants homozygous for the
construct did not contain any white seeds, indicating a successful rescue of the embryo-
defectiveness of mkip1-/- (Fig. 1a, Supplementary Fig. 1d). We recovered rescued seedlings in
both mkip1-1-/- and mkip1-2-/- backgrounds at the expected frequency of ~25%. However, after
germination, these seedlings developed serrated, white true leaves and abnormal meristems
and were seedling lethal (Fig. 1c, Supplementary Fig. 1e). These phenotypes were similar to
ABI3-rescue lines of mutants severely impaired in chloroplast differentiation due to primary
defects in plastidial translation or splicing29. Seedling lethality of rescued mkip1-1-/- could not be
rescued by growing plants on ½-strength MS medium supplemented with 1% sucrose
(Supplementary Fig. 1c).

Plastids from true leaves of ABI3-rescued mkip1-1-/- seedlings mostly contained collapsed
thylakoid stacks and accumulated electron-dense, vesicular structures of unknown nature.
Occasionally, we observed chloroplast-like plastids with large condensed thylakoid stacks (Fig.
1e, outer right micrograph), reminiscent of structures observed upon co-suppression of the
plastidial ribosomal protein AtbS1c (RLPS1)30. The cotyledons were pale green, and their plastids
had abnormally stacked thylakoids (Fig. 1c, e). Presumably, the cotyledons were less affected
due to residual AtMKIP1-mCitrine protein remaining in cotyledon tissue from embryogenesis,
although AtMKIP1-mCitrine levels were below the detection limit (Fig. 1d). These phenotypes
strongly suggest that AtMKIP1 is essential for plant viability also after embryogenesis and are
consistent with a putative essential role of AtMKIP1 in chloroplast function, but not in starch
metabolism.

AtMKIP1 and orthologs have lost branching enzyme activity
Phylogenetic analyses showed that the canonical BEs involved in starch biosynthesis of land
plants and streptophyte algae belong to either class I (which is absent in Arabidopsis) or class II
(to which AtBE2 and AtBE3 belong; Fig. 2a), confirming previous observations31. The BEs of
chlorophyte algae also mostly belong to these classes, whereas those of glaucophyte and
rhodophyte algae, which produce floridean starch in the cytosol32, form a separate group.
AtMKIP1 and its orthologs form yet another, well-separated clade of class III BEs31, which share
the structural features of AtMKIP1 described below. We identified at least one class III BE in
each photoautotrophic land plant examined and in some streptophyte algae, but not in any other
alga (Fig. 2a, Supplementary Fig. 4a), suggesting that it arose during the colonization of land
by plants (Fig. 2a, Supplementary Fig. 4a). The origin of class III BEs is unclear, but their
distinct exon-intron gene organization31 (Supplementary Fig. 4b), renders it unlikely that they
arose from the duplication of a class I or II BE gene.

Class III BEs retain a classical BE domain architecture consisting of a family 48 carbohydrate-
binding module (CBM) and α-amylase domains (Fig. 2b). Nonetheless, they display striking
differences from canonical BEs, with which they share only ~30% amino acid (AA) identity31.
First, class III BEs possess an MKIP1-specific region (MSR) of ~150 AA after the CBM. This
stretch is enriched for positively and negatively charged AA (especially lysines and glutamates,
respectively) and is predicted to form a largely disordered region protruding from the AtMKIP1
core (Supplementary Fig. 5). An AtMKIP1(ΔMSR)-YFP version lacking the MSR failed to
complement the embryo defects of mkip1-1-/-, despite being expressed as a soluble protein,
indicating a key role for AtMKIP1 function (Supplementary Fig. 6).

Class III BEs also carry substitutions in several residues required for starch branching catalysis,
including two aspartate residues of the catalytic triad33 (H558 and N682 in AtMKIP1; Fig. 2b),
consistent with AtMKIP1’s apparent absence of BE activity23,25. To test whether glucan BE
activity is also absent in orthologous proteins, we expressed MKIP1 versions from tobacco
(Nicotiana tabacum), maize (Zea mays), a moss (Physcomitrium patens) and a streptophyte
alga (Coleochaete scutata) as YFP-tagged proteins in the yeast Saccharomyces cerevisiae
(Fig. 2c). The parental yeast strain used, “578”, contains all components required for glucan
production except a BE, as it has non-yeast genes encoding a starch synthase (AtSS1) and an
enzyme producing its substrate ADPglucose (EcglgC-TM-HA), but lacks its endogenous
glycogen BE as well as all other yeast glycogen-metabolic genes34. All class III BEs were
expressed as soluble proteins in yeast, although in some cases at low levels (Fig. 2d).
However, none of them showed enzymatic activity on native BE-activity gels, whereas all tested
class I/II BEs did (Fig. 2e). Expression of class III BEs also did not result in glucan synthesis in
yeast, as judged by the absence of dark staining after iodine staining of glucans produced in
yeast cells (Fig. 2f). The BE activity of AtMKIP1-YFP could not be restored by reconstituting its
former catalytic triad in the AtMKIP1(H558D,N682D)-YFP version (Fig. 2e, f). These data
suggest that the absence of BE activity is a general feature of class III BEs and is likely due to
widespread sequence divergence.

To test whether the remaining BE-like features in AtMPKIP1 are nevertheless required for its
function in Arabidopsis, we mutated a glutamate and a histidine residue essential for canonical
BE activity as acid/base catalyst35 and transition-state stabilizer36, respectively. Expression of
this AtMKIP1(E612A,H681A)-mCitrine construct complemented the mkip1-1-/- mutant phenotype
similarly to the non-mutated AtMIKIP1-mCitrine construct (Supplementary Fig. 2).
Complementation was also observed by AtMKIP1(W151A,W171A)-mCitrine (Supplementary
Fig. 2), which carries substitutions in conserved tryptophan residues critical to glucan binding of
family-48 CBMs37,38. Taken together, these data demonstrate that AtMKIP1 function
predominantly requires others features than canonical BEs, including its newly evolved MSR
domain.

MKIP1 interacts with MatK in an RNA-independent manner
We conducted co-immunoprecipitation (IP) experiments coupled with mass spectrometry (MS)
to identify potential interaction partners of AtMKIP1. AtMKIP1-YFP stably expressed in
Arabidopsis significantly enriched AtMatK in three independent anti-YFP IPs, each with multiple
replicates (Fig. 3a). These experiments further suggested that AtMKIP1 interacts with two other
plastid-localized proteins essential for plant viability: AtValRS2 (AtEMB2247), a putative valyl-
tRNA synthetase39, and AtEMB3120 (EMBRYO-DEFECTIVE 3120 or SNOW-WHITE LEAF1,
SWL1), which is a stromal protein required for plastid development, but of unknown molecular
function40,41. We confirmed the interactions between AtMKIP1, AtValRS2 and AtEMB3120 by
multiple (reverse-)IPs performed in Arabidopsis (Fig. 3b, Supplementary Fig. 7). However, as
the functions of AtValRS2 and AtEMB3120 are not or only poorly characterized, we focus here
on AtMatK.

Since AtMatK is encoded in the plastome, it cannot be routinely manipulated in Arabidopsis, and
our attempts to raise a working antibody against AtMatK were unsuccessful. We thus used
NtMatK C+ transplastomic tobacco (N. tabacum), which expresses NtMatK with a C-terminal
triple HA tag at its native plastome locus12 (Fig. 3c). The NtMatK-HA protein is functional since it
can substitute for NtMatK, which is considered an essential protein12. In IP experiments with
tobacco seedlings, NtMKIP1 co-precipitated with NtMatK-HA (line NtMatK C+; Fig. 3d). This
interaction was not abolished by treating the IP samples with an RNase A/T1 mixture, which
effectively degraded the RNA (Supplementary Fig. 8), suggesting that the interaction is not
bridged by (intron) RNA (Fig. 3d). MS analysis of an independently performed IP experiment
showed a >60-fold and statistically highly significant enrichment of the bait NtMatK-HA and of
the tobacco orthologs of AtMKIP1, AtValRS2 and AtEMB3120 (Fig. 3e). In addition, NtRNC1
and a CRM (chloroplast RNA splicing and ribosome maturation) domain protein, both homologs
of plastidial splicing factors from maize or Arabidopsis21,42, were significantly, albeit weakly
enriched in these IPs. Several plastidial chaperonins were also enriched (e.g., chaperonins-60 α
and β; Supplementary Table 1), possibly indicating a need for folding assistance for NtMatK-
HA complex formation. However, NtRNC1, the CRM domain protein and chaperonins were only
enriched 2 to 6-fold, suggesting a relatively loose and/or transient association with NtMatK-HA.
Furthermore, none of them were enriched in the Arabidopsis IP-MS experiments (Fig. 3a).

To investigate if the MKIP1 interactors together form one complex, we analyzed chloroplast
stromal extracts from NtMatK C+ tobacco and wild-type Arabidopsis plants using sucrose
density gradient centrifugation and size-exclusion chromatography (SEC). In SEC of NtMatK C+
stromal extracts, NtMatK-HA, NtMKIP1, and NtValRS2 co-migrated in high-molecular weight
fractions (fraction ≤8), with some NtMKIP1 and NtValRS2 also present in lower molecular
weight fractions (Fig. 4a). Since the anti-HA antibody cross-reacted with Rubisco large subunit
(NtrbcL), among others, we purified NtMatK-HA and its interactors by anti-HA IP from all
fractions. This confirmed the presence of NtMatK-HA, NtMKIP1, and NtValRS2 in high-
molecular weight fractions (Fig. 4b). NtEMB3120 could not be detected by immunoblotting.
However, proteomic MS/MS analysis of the unpurified SEC fractions 5 and 6, containing the
highest amounts of NtMKIP1 and NtMatK, showed that NtEMB3120 was also present in these
high-molecular weight fractions (Fig. 4c).

On sucrose gradients, NtMatK-HA, NtMKIP1, and NtValRS2 again migrated into high-molecular
weight fractions (Supplementary Fig. 9a). Similarly, when assessing extracts from Arabidopsis,
AtMKIP1 and AtValRS2 co-migrated in high-molecular weight fractions of sucrose gradients
(AtMatK and AtEMB3120 could not be detected by immunoblotting; Supplementary Fig. 9b).
Interestingly, the migration of AtMKIP1 and AtValRS2 did not change upon treatment of extracts
with RNase, indicating that the formation of a high-molecular weight MKIP1 complex does not
depend on intact RNA (Supplementary Fig. 9b).

To gain insight into the complex’ stoichiometry, we tested the subunits for potential self-
association. No self-association was observed for AtMKIP1, AtValRS2 and AtEMB3120 in IP
experiments after transient expression in N. benthamiana (Supplementary Fig. 10a-c). We
were not successful in expressing AtMatK in this system, nor stably in Arabidopsis, despite
using a version codon-optimized for the Arabidopsis nuclear genome. However, we could
express AtMatK with epitope tags as a soluble protein in yeast, allowing us to conduct IP
experiments in this system. As for the other subunits, AtMatK did not efficiently interact with
itself, while it did interact with AtMKIP1 (Supplementary Fig. 10d). Together, our data indicate
that the four subunits together form a stable complex. This is consistent with the complex model
from AlphaFold3, which predicts a four-subunit heteromultimer of AtMKIP1, AtMatK, AtValRS2
and AtEMB3120 with a 1:1:1:1 stoichiometry with intermediate confidence (Fig. 5). AlphaFold3
did not support homo-dimerization of either component, nor addition of a second copy of any of
the four proteins (Supplementary Fig. 10e).

AlphaFold3 further confidently predicted that AtMKIP1 and AtMatK directly interact via a large
interaction platform formed by the CBM-MSR region of AtMKIP1 and the N-terminal region of
AtMatK (Fig. 6a-d). To test a direct interaction, we co-expressed AtMatK-HA with AtMKIP1-YFP
or AtBE3-YFP as control for IP experiments in yeast. As predicted, the bait AtMKIP1-YFP
efficiently co-precipitated AtMatK-HA (strain D), whereas AtBE3-YFP did not (strain C; Fig. 6e).
In reciprocal interaction assays, AtMatK-HA efficiently co-precipitated AtMKIP1-YFP, but not
AtBE3-YFP (Supplementary Fig. 11b). These results confirm a direct and specific interaction
between AtMKIP1 and AtMatK. We next tested the involvement of the CBM-MSR region of
AtMKIP1 in this interaction (Fig. 6e). An AtMKIP1-YFP variant without MSR (ΔMSR; strain E)
co-precipitated much less AtMatK-HA than the entire AtMKIP1-YFP (strain D) in yeast. In turn,
MSR-YFP co-precipitated AtMatK-HA (strain F) only slightly less efficiently than the entire
AtMKIP1-YFP (strain D). When testing an AtMKIP1 segment comprising its CBM and MSR, little
YFP-tagged protein was expressed and recovered in the anti-YFP IP (strain G) for reasons
unknown. The precipitated CBM-MSR-YFP protein, however, efficiently co-precipitated AtMatK-
HA. These results indicate a key role of the CBM-MSR region of AtMKIP1 in interacting with
AtMatK.

Equivalent IP experiments using AtMatK-HA segments (Fig. 6f, g) showed that the N-terminal
region of AtMatK-HA co-precipitated well with the whole AtMKIP1 (strain I) and the CBM-MSR
region of AtMKIP1 (strain J), whereas the C-terminal X domain of AtMatK-HA did not (strains M
and N). Similarly, in reciprocal IPs using AtMatK-HA as bait, its N-terminal portion but not the C-
terminal X domain strongly co-precipitated AtMKIP1-YFP (Supplementary Fig. 11).
Collectively, these data suggest that the CBM-MSR of AtMKIP1 and the N-terminal region of
AtMatK form an interaction platform. Contrarily, AtMatK’s X domain likely contributes little or not
at all to the protein-protein interactions with AtMKIP1 and – according to the AlphaFold3
complex prediction – also to the other complex subunits (Fig. 5b).

AtMKIP1 is associated with group IIA introns in vivo
We conducted RNA co-IP coupled with RNA sequencing to identify RNA regions specifically
associated with AtMKIP1 or its AtMatK complex. We therefore immunoprecipitated RNA
associated with AtMKIP1-YFP in Arabidopsis seedlings, using a line expressing AtBE3-YFP as
control. Immunoblot analysis showed the binding of AtMKIP1’s interaction partners, confirming
the success of the IP (Fig. 3b). Analysis of the RNA segments that were differentially enriched
showed that AtMKIP1-YFP effectively co-precipitated the RNAs of all group IIA intron containing
genes previously identified to be associated with NtMatK-HA12,13 (Fig. 7a). This enrichment was
not due to greater abundance of these RNAs in the AtMKIP1-YFP expressing line, as they were
not or only slightly more abundant (<2-fold) in the input RNA fraction of this line. AtMKIP1-YFP
also co-precipitated RNAs of the intron-free genes rpl23 and rps19, which are co-transcribed
within the rpl2-containing operon43. However, while rpl2 RNA was markedly and significantly
enriched throughout the intron and exons, only short segments (< 200 nt) of rpl23 and rps19
flanking rpl2 were >4-fold enriched, suggesting co-enrichment with rpl2 (Fig. 7b). The lack of
co-enrichment of distal transcript segments is likely due to the nuclease activities present in the
chloroplast stroma and during the IP bead incubation step, which lead to RNA fragmentation
except for regions protected by proteins. This enables the specific recovery of the RNA regions
that are directly or indirectly (e.g., via a protein or RNA bridge) associated with the bait
protein12,13.

Fine-mapping of the other RNA regions co-precipitated with AtMKIP1-YFP showed that trnK
enrichment peaked at the junction between intron domains III and IV, just upstream of the matK
open-reading frame (ORF) (Fig. 7b). The exons and most other trnK intron regions were also
enriched, overall largely resembling the enrichment observed by RNA co-IP of NtMatK-HA12,13. The remaining binding targets shared a strong enrichment of intron domain II. For rps12 intron
2, enrichment was low and domain II was the only intron domain significantly enriched,
contrasting the pattern observed for NtMatK-HA13. However, for most other targets (atpF, rpl2,
trnI, and trnA), strong enrichment was also observed in intron domains IV and VI. In the case of
trnV, enrichment was relatively low, consistent with its variable enrichment by NtMatK-HA12,13. In
addition, domain VI of trnV was not enriched; this domain may be minimally involved in intron
splicing of trnV because it lacks the bulged adenosine normally used in the first splicing step15.
Overall, these results show a remarkable overlap of the intron regions associated with AtMKIP1-
YFP and those previously identified for NtMatK-HA12,13, suggesting a close functional link
between MKIP1 and MatK.

AtMKIP1 facilitates splicing of plastidial group IIA introns
The deficiency of AtMKIP1 beyond the cotyledon stage in the ABI3-rescue plants led to seedling
lethality, limiting the material available for splicing analyses. The likely pleiotropic defects of
these lines also make identifying the primary defect difficult, since splicing and translation in
plastids are linked. Because group IIA introns disrupt tRNAs and transcripts of ribosomal
proteins, severe splicing defects will entail defects in translation. In turn, severe plastidial
translation defects can reduce the abundance of plastome-encoded MatK, thereby indirectly
affecting group IIA intron splicing44,45. To catch the primary defect of AtMKIP1 loss, we inducibly
downregulated AtMKIP1 at later developmental stages using two estradiol-inducible artificial
microRNA silencing lines in Arabidopsis, each targeting a different site of the AtMKIP1 mRNA
(amiR-mkip1 plants). Additionally, we created an equivalent line targeting AtuL4c mRNA, which
encodes an essential protein of the plastidial large ribosomal subunit27,46. This amiR-uL4c line
was used as a control for splicing defects caused by a plastidial translation defect itself.
Estradiol treatment of amiR-uL4c plants resulted in pale newly developed leaves, especially at
their base, where the tissue is the youngest and chloroplasts are still differentiating (Fig. 8a).
Chlorosis was milder in treated amiR-mkip1 plants but also apparent, and chloroplasts from pale
amiR-mkip1-1 leaves had abnormally few thylakoid membranes (Supplementary Fig. 12). We
focused on newly developed leaves (red arrows in Fig. 8a) in downstream molecular analyses.
Protein quantification by immunoblotting and proteomics (Fig. 8c, d) showed that amiR-uL4c
and amiR-mkip1 tissues were strongly depleted of the respective proteins targeted by silencing.
However, all silencing lines had higher levels of AtValRS2 and AtEMB3120, and estradiol-
treated amiR-uL4c leaves additionally had higher levels of AtMKIP1. These alterations are
consistent with the putative roles of AtMKIP1, AtValRS2 and AtEMB3120 in plastid gene
expression and the increased expression of such genes when chloroplast differentiation is
delayed47.

We next assessed the newly developed leaves for defects in plastidial translation. In estradiol-
treated amiR-uL4c leaves, we observed a significant reduction of almost all quantified plastome-
encoded proteins, indicating a defect in plastidial translation (Fig. 8b). This tissue was depleted
in plastidial ribosomes, as the levels of plastidial rRNAs (Fig. 8e, f) and of ribosomal proteins
(Supplementary Fig. 13c) were strongly reduced. We also detected plastidial rRNA processing
intermediates (Supplementary Fig. 14); this is consistent with the view that some rRNA
processing steps take place after rRNA incorporation into the ribosomal subunit(s),
necessitating enough pre-ribosomes and thus plastidial translation for normal rRNA
processing1,48–51.

Defects in plastidial translation, ribosome accumulation and rRNA processing were also
apparent upon silencing of AtMKIP1 but in a considerably milder form (Fig. 8, Supplementary
Figs. 13c and 14). Silenced amiR-mkip1 leaves showed no significant change in AtMatK
abundance (Fig. 8d). Milder pleiotropic defects were also evident when comparing all 445
differentially abundant proteins (compared to the WT) among the 6889 quantified proteins,
where changes were generally strongest in amiR-uL4c and mildest in amiR-mkip1-2 plants
(Supplementary Fig. 13).

We next assessed the efficiency of intron splicing in newly emerged, silenced tissue. We
therefore quantified the relative ratios of spliced to unspliced plastid mRNAs using reverse
transcription quantitative real-time PCR (RT-qPCR)52,53. Given the difficulties associated with
reverse transcription of tRNAs, their splicing was determined by northern blotting, circumventing
cDNA synthesis. Splicing of the group IIB intron in rps16 was not assessed, since this is a
pseudogene in Arabidopsis with no apparent splicing even in the WT54,55.

No significant splicing defects compared to WT were observed in mock-treated plants of any
amiR lines (Fig. 9b, Supplementary Figs. 15 and 16). However, upon estradiol-induced
silencing, young amiR-mkip1 tissue showed major splicing defects in most of the introns
identified to be associated with AtMKIP1-YFP. The introns of trnK, trnA, rps12 (intron 2), rpl2,
and atpF showed severely reduced splicing efficiency (i.e., a lower ratio of spliced to unspliced
RNA) and less absolute spliced RNA compared to estradiol-treated WT and amiR-uL4c plants
(Fig. 9, Supplementary Fig. 15). Mild splicing defects in AtMKIP1 silenced tissue were also
observed for trnI and trnV (Fig. 9b, c). In AtuL4c silenced tissue, the MatK binding targets
showed only little or no impaired splicing (Fig. 9, Supplementary Fig. 15). Despite the slight
reduction in AtMatK (-29 ± 3%; Fig. 8d), its abundance thus seemed still largely sufficient for
intron splicing. This is in agreement with a previous report of Arabidopsis that MatK targets are
correctly spliced despite impaired plastid translation29.

The ratio of spliced to unspliced RNA upon AtMKIP1 silencing was also somewhat reduced in
several introns not associated to AtMKIP1-YFP (Fig. 9e). However, in all cases other than the
petB, petD and ndhA intron, this was due to the accumulation of the unspliced precursor, while
the spliced RNA appeared not to be reduced compared to WT (Supplementary Fig. 15). In
addition, here AtuL4c silencing caused analogous defects, suggesting that these are secondary
effects associated with chloroplast malfunction2. Collectively, these findings strongly suggest
that AtMKIP1 silencing causes a specific and primary splicing defect in all introns associated
with MatK and MKIP1. These defects likely underlie the observed plastidial translation defects,
ultimately leading to pale leaves in the amiR-mkip1 plants, and, in the mkip1-/- lines, to the early
embryonic defects.

Methods
Mutant lines and generation of transgenic plants
Unless otherwise indicated in the figures, experiments were performed on Arabidopsis single-
insertion lines of the T3 or a later generation. Where zygosity is not indicated in the Figures,
plants were either homo- or heterozygous for the insert. Binary plant expression constructs were
transformed into Agrobacterium tumefaciens (strain GV3101). Arabidopsis thaliana (Col-0)
plants were transformed by floral dipping. Transformed T1 plants were selected for their
resistance to hygromycin or Basta on 0.8% (w/v) agar plates containing one-half-strength
Murashige and Skoog (MS) medium including vitamins (Duchefa Biochemie) at pH 5.7 with 25
mg l-1 hygromycin B or 15 mg l-1 glufosinate ammonium (BASTA; from Merck) and further tested
for transgene expression by immunoblotting. Single insertion lines were identified based on the
segregation ratio of resistant to non-resistant plants in the T2 generation.

T-DNA insertion lines were in Col-0 background from the SALK collection and were obtained
from the European Arabidopsis Stock Centre under the following identifiers: SALKseq_040659
(mkip1-1), SALK_129083 (mkip1-2 or emb2729-2) and SALK_070161 (valrs2-1). Primers used
for genotyping are listed in Supplementary Data 1. Insertion sites were confirmed by Sanger
sequencing of the PCR amplicon containing the border between the T-DNA and its genomic
insertion site.

Homoplastomic Nicotiana tabacum (tobacco) lines modified to express NtMatK fused to a C-
terminal 3x HA tag (line NtMatK C+) or control lines carrying only the selection marker (NtMatK
C-)12 were kindly provided by Christian Schmitz-Linneweber (HU Berlin, Germany).

Cloning of plant constructs
Unless otherwise noted, all plasmids for protein expression in Arabidopsis were generated using
Gateway recombination cloning technology (Invitrogen). The plasmids were validated by
sequencing of the insert(s) (when PCR-amplified fragments were cloned) and/or diagnostic
restriction digests (when PCR was not used).

For AtMKIP1, the CDS of splice form 2 (AT3G20440.2) was considered the primary splice form
based on the mapping coverage of available RNA- sequencing data at J Browse69. The CDS of
the full-length primary splice forms of AtMKIPI1 (AT3G20440.2), AtEMB3120 (AT3G14900.1),
and AtValRS2 (AT5G16715.1) flanked by attB sites were amplified by PCR from Arabidopsis
Col-0 cDNA and individually recombined into pDONR221. The resulting vectors
(AtMKIP1_pDONR221, AtEMB3120_pDONR221, and AtValRS2_pDONR221) were separately
recombined with pUBC-YFP70, which allows UBQ10-driven expression of proteins carrying a C-
terminal YFP tag. For 35S-driven expression of AtMKIP1 with a C-terminal YFP tag,
AtMKIP1_pDONR221 was recombined with the gateway vector pB7WG2.

For expression of AtMKIP1’s CDS with a C-terminal mCitrine tag driven by PABI3 or PUBQ10, we
first amplified the respective promoter sequences (for PABI3: 2.2 kb preceding the start codon of
ABI3, AT3G24650; for PUBQ10: the 0.6 kb promoter as in pUBC-YFP) flanked by attB sites by
PCR using Arabidopsis Col-0 genomic DNA or the pUBC-YFP vector as templates. After
recombining the PCR products into pDONR P4-P1R, the resulting promoter vectors
(PABI3_pDONRP4-P1R and PUBQ10_pDONRP4-P1R) were separately recombined with
mCitrine_pDONR P2R-P3 (ref. 38) and AtMKIP1_pDONR221 into the gateway multi-site
destination vector pB7m34GW2.

For expression of the CDS of AtMKIP1, AtValRS2, and AtEMB3120 with a C-terminal 3x FLAG
tag driven by PUBQ10, the vectors AtMKIP1_pDONR221, AtEMB3120_pDONR221, and
AtValRS2_pDONR221 were individually recombined with PUBQ10_pDONRP4-P1R and
3xFLAG_pDONR P2R-P3 (containing a 3x FLAG tag, followed by a stop codon) into
pB7m34GW2.

Point mutations in the CBM of AtMKIP1 (W151A, W171A) or the central α-amylase domain of
AtMKIP1 (E612A, H681A) were introduced into AtMKIP1_pDONR221 using two sequential
rounds of site-directed mutagenesis for each substitution (QuikChange; Agilent Technologies).
The resulting AtMKIP1(W151A,W171A)_pDONR221 and AtMKIP1(E612A,H681A)_pDONR221
vectors were then separately recombined with PUBQ10_pDONRP4-P1R and mCitrine_pDONR
P2R-P3 (ref. 38) into pB7m34GW2. To express a ΔMSR version of AtMKIP1, the MSR (AA 178-
333 of AtMKIP1) in AtMKIP1_pUBC-YFP was deleted using NEBuilder HiFi DNA Assembly
(New England Biolabs) by fusing PCR-amplified vector fragments covering the entire vector
except for the MSR, resulting in pBP605_AtMKIP1_deltaMSR(178-333)_pUBC-YFP.

amiRNAs (21mers; Supplementary Data 1) for inducible silencing of AtMKIP1 or AtuL4c were
designed using the WDM3 MicroRNA Designer tool. Synthesized amiRs in the MIR319a
backbone flanked by attB sites were first recombined into pDONR221 and then into the binary
pH7m34GW vector71 containing an estrogen-inducible XVE cassette driven by PUBQ10.

Plant growth and β-estradiol treatment
Arabidopsis and tobacco plants were grown in Percival AR-95L chambers (CLF Plant Climatics)
under 12-h light / 12-h dark cycles as previously described72. For β-estradiol-induced silencing,
3-week old plants were sprayed once per day with aqueous β-estradiol solution (20 µM β-
estradiol [Sigma-Aldrich], 0.2% [v/v] dimethyl sulfoxide [DMSO], 0.01% [v/v] Silwet L-77). The
mock spray solution was prepared with the same composition but omitting β-estradiol. Newly
emerged leaves after 11 days of treatment were flash-frozen in liquid N2 and stored at -80°C
until further processing. Each replicate derives from an individual plant.

Proteome analysis of β-estradiol treated plants
Frozen leaf material of was ground by mortar and pestle in liquid N2. About 15 mg of plant
material was subjected to peptide preparation using the iST kit for plant tissues (PreOmics,
Germany) following the manufacturer’s instructions. Dried peptides were re-solubilized by
sonication in 3% (v/v) acetonitrile, 0.1% (v/v) formic acid. Peptides were spiked with iRT
peptides (Biognosys) and analyzed on Orbitrap Exploris 480 mass spectrometer (Thermo Fisher
Scientific) equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific) and coupled
to an M-Class UPLC (Waters) equipped with a nanoEase M/Z Symmetry C18 trap column (100
Å, 5 µm, 180 µm x 20 mm, Waters) and a nanoEase M/Z HSS C18 T3 analytical column (100 Å,
1.8 µm, 75 µm x 250 mm, Waters).

Peptides were separated at a flow rate of 300 nl min-1 by the following 90-min gradient: initial
hold at 5% solvent B (0.1% formic acid in acetonitrile) / 95% solvent A (0.1% formic acid in
water) for 3 min, followed by a gradient to 25% B / 75% A in 80 min, and to 32% B / 68% A in 10
min, a cleaning step at 95% B for 10 min and re-equilibration at 5% B / 95% A for 10 min (all
percentages v/v). MS data were acquired in data-independent acquisition (DIA) mode using the
following settings: The full MS was scanned at a resolution of 60,000 with a normalized
automatic gain control (AGC) target value of 300% and a maximum injection time of 45 ms. The
mass range was set to 400–1000 m/z. Peptides were fragmented at a normalized collision
energy of high-energy collision dissociation (HCD) of 30%. The normalized AGC target value for
fragment spectra was set to 1000%. At the MS2 level, 60 windows of 10 m/z with optimized
window placement and without overlap within the mass range of 400–1000 m/z were analyzed
at a resolution of 30,000. Maximum injection time mode was set to automatic.

In addition, two pools composed of one replicate of each group were analyzed by data-
dependent acquisition (DDA) for subsequent spectral library built, using the same gradient as
for DIA and the following settings: full MS scan with a resolution of 120,000 with a normalized
AGS target of 300%, a maximum injection time of 45 ms, and a mass range of 350-1200 m/z.
Precursors were filtered for charge states between 2 and 7, selected for MS/MS based on a
minimum intensity of 5,000 and fragmented by HCD using a normalized collision energy of 30%.
Fragment spectra acquired with a resolution of 15,000. Precursor masses previously selected
for MS/MS measurement were excluded from further selection for 20 s, and the exclusion
window was set to 10 ppm.

DIA-MS raw files were converted into mzML files using MSConvert73 (v3.0.2472-63d00b1). The
MS data were further processed using MSFragger-DIA implemented in the computational
platform FragPipe74 (v23.1) with DIA-NN75 (v2.1.0). A hybrid spectral library was built from the
primary DIA data and the auxiliary DDA data using the DIA_SpecLib_Quant workflow with
default settings. Spectra were searched with MSFragger76 (v4.3) against the Araport 11
proteome database concatenated to its reversed decoyed fasta database and common protein
contaminants using default settings, i.e. allowing for one missed tryptic cleavage and setting
carbamidomethylation of cysteine as fixed modification and methionine oxidation and
acetylation of the protein N-terminus after methionine removal as variable modifications. DIA
data was analyzed with the hybrid spectral library as input and using the same FragPipe
workflow and the same search settings against the Araport 11 database as above. Average log2
fold changes (FC) and adjusted p-values were calculated in Amica77 (v3.0.1) webserver using
the DIA workflow, filtering for proteins with at least 3 valid values in each group, setting re-
normalization (in addition to that previously conducted by DIA-NN) and imputation of missing
values to “none”, and using “limma” for differential expression analysis. Only proteins that were
identified by at least two peptides, with at least one of them being uniquely assigned, were kept
for further data analysis. In the three cases where two individual splice-form variants had been
quantified separately due to the detection of splice-form specific peptides, the variant with fewer
quantified peptides was excluded from the analysis.

For proteins that were strongly and statistically significantly changed in at least one line
compared to the wild-type control (i.e. log2 FC <|1|, adjusted p-value <0.05), the number of
appropriate clusters was identified by the Elbow method using the “wss” function in RStudio
(v2024.12.1), followed by grouping the proteins into 4 clusters by k-means clustering using the
“kmeans” function. Corresponding genes that are overrepresented in each cluster compared to
those corresponding to the in total 6,688 proteins quantified by proteomics were identified by
PANTHER78 overrepresentation tests (Released 20240807) using Fisher as test type,
Bonferroni method for p-values corrected for multiple comparison, and the GO database
released 25200316 (DOI: 10.5281/zenodo.15066566) for gene annotation. Protein locations are
the predicted/annotated consensus locations retrieved from SUBA5 (Ref. 79). Localization
enrichments in each cluster were calculated compared to the localizations of the total 6688
proteins quantified by proteomics. In case of multiple locations, only the first one was
considered, since multiple locations in the four protein clusters was deemed negligible (~1%).
Plastidial ribosomal proteins were annotated following the nomenclature from Scarpin et al.80.

Plant imaging
For imaging Arabidopsis chloroplasts by transmission electron microscopy (TEM), leaf sections
were fixed, embedded, and imaged as described previously72.

For observing AtMKIP1-YFP localization and co-localisation with either mitochondria or nuclei,
rosette leaves from 4-week-old, soil-grown Arabidopsis plants were syringe-infiltrated with
infiltration medium (10 mM MES-KOH, pH 5.6, 10 mM MgCl₂) containing 100 nM MitoTracker
Red CMXRos (from a 1 mM stock in 100% DMSO) or 10 µg ml-1 DAPI (dissolved in water). The
abaxial leaf sides were imaged using a Zeiss LSM880 confocal microscope equipped with 40×
water objective. Excitation was performed with the 405-nm diode (for DAPI), the 514-nm Argon
laser (for YFP), the 561-nm Argon laser for MitoTracker, and the 633-nm helium-neon laser
(chlorophyll). The emitted signals were detected at 410–511 nm for DAPI, 519–566 nm for YFP,
578–643 nm for MitoTracker, and 647–721 nm for chlorophyll. Image acquisition and processing
were performed using ZEN Microscopy Software.

To image Arabidopsis embryos, seeds dissected from a silique were collected in a
microcentrifuge tube and incubated in a 1:1 mixture of ethanol and acetic acid for 1 h at 20°C
with rotation, then placed on a microscope glass slide and further cleared by incubation with
Hoyer’s medium (7.5 g gum arabic, 100 g chloral hydrate, 5 ml glycerol and 30 ml water) for 1 h.
Cleared seeds were imaged using a Leica DM2500 microscope.

Plant protein extraction and immunoblotting
Total plant protein extraction from leaf disks and immunoblotting using IRDye 800CW goat anti-
rabbit IgG secondary antibody (LICORbio) were conducted as described previously72. Total
proteins on membranes were stained with Revert 700 (LICORbio). Antibody sources, dilutions
and validation are detailed in Supplementary Data 1.

Immunoprecipitation in plants for immunoblot detection
For anti-YFP IP experiments in Arabidopsis, freshly harvested plant tissue was homogenized in
ice-cold extraction buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 1% [v/v] Triton X-
100, 1x protease inhibitor cocktail [cOmplete EDTA-free, Roche]) using equal ratios of buffer to
input material for all samples and a Dounce homogenizer. After centrifugation at 19,000 g and
4°C for 5 min, the supernatant containing soluble protein was incubated with pre-equilibrated
magnetic agarose anti-YFP beads (GFP-Trap, ChromoTek; 10 µl beads per 0.1 g plant material)
for 1 h at 4°C with rotation, washed three times with ice-cold wash buffer 1 (same as extraction
buffer, but replacing Triton X-100 with 0.5% [v/v] NP-40), followed by three washes with ice-cold
wash buffer 2 (50 mM Tris–HCl, pH 8.0, 150 mM NaCl), each time collecting the beads on a
magnetic rack. In the last wash step, the resuspended beads were transferred to a fresh tube to
remove contaminants sticking to the tube. Proteins bound to the beads after washing were
eluted by boiling the beads in SDS-PAGE loading dye (50 mM Tris-HCl, pH 6.8, 2% [w/v] SDS,
100 mM DTT, 3% [v/v] glycerol, and 0.005% [w/v] bromophenol blue) at 95°C for 5 min. Input
samples are aliquots of soluble proteins not subjected to immunoprecipitation.

For the anti-HA IP in N. tabacum, 10-day-old seedlings were used as the starting material.
Soluble protein extraction and bead incubation were carried out as described above for
Arabidopsis samples but using anti-HA beads (Anti-HA high affinity, Roche). After protein
binding during the incubation step, the beads were collected by centrifugation at 13,000 g for 10
s, then washed three times with extraction buffer and three times with dilution buffer (50 mM
Tris–HCl, pH 8.0, 150 mM NaCl). In the final wash step, the resuspended beads were
transferred to a new tube and collected by centrifugation. Bound proteins were eluted as for
Arabidopsis. If samples were RNase treated, RNase A/T1 mix (Thermo Fisher) was added to
the soluble protein extracts (final concentrations: 100 µg ml-1 for RNase A and 250 U ml-1 for
RNase T1) together with the anti-HA beads.

Transient protein expression in tobacco and subsequent IP experiments using magnetic
agarose anti-YFP beads (GFP-Trap, ChromoTek) were conducted as described81, except that
wash buffer 1 (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM DTT and 0.5% [v/v] NP-40) was
used for all five washes. The p19 silencing suppressor was co-expressed in all cases82. Each
replicate derives from a separate infiltrated plant.

Immunoprecipitation in plants followed by mass spectrometry
For IP-MS experiments in Arabidopsis, we followed a procedure previously described38 using
anti-YFP µMACS magnetic beads (Miltenyi Biotec) and protein identification on a Q Exactive
mass spectrometer (Thermo Fisher Scientific) with the following modifications: 1) proteins eluted
from the beads were precipitated in 20% (w/v) trichloroacetic acid; 2) when needed, peptides
were cleaned using the Phoenix kit (PreOmics) following the manufacturer’s instructions; 3)
dried peptides were solubilized in 3% (v/v) acetonitrile, 0.1% (v/v) formic acid prior to MS
analysis; 4) liquid chromatography of peptide separation was conducted at a flow rate of 300 nl
min-1 by a gradient from 5% solvent B (0.1% formic acid in acetonitrile) / 95% solvent A (0.1%
formic acid in water) to 35% B / 65% A in 90 min, to 60% B / 40% A in 5 min and to 80% B /
20% A in 1 min.

The acquired raw MS data were processed by MaxQuant83 (v1.6.2.3), followed by protein
identification using the integrated Andromeda search engine. A separate MaxQuant analysis
was performed for each of the three IP experiments. Spectra were searched against the Araport
11 database (v2017-02-01), concatenated to its reversed decoyed FASTA database and
common protein contaminants, setting cysteine carbamidomethylation as fixed and methionine
oxidation and N-terminal protein acetylation as variable modifications, enzyme specificity to
trypsin and allowing a maximum of two missed-cleavages, and using MaxQuant Orbitrap default
search settings. The maximum false discovery rate was set to 0.01 for peptides and 0.05 for
proteins, label free quantification enabled and a 2-min window for matching between runs
applied. Each file was kept separate in the MaxQuant experimental design to obtain individual
quantitative values. Fold changes of protein abundances were computed based on intensity
values reported in the proteinGroups.txt file. A set of functions implemented in the R package
SRMService was used to filter for proteins with 2 or more peptides allowing for a maximum of
up to 3 missing values, to normalize the data with a modified robust z-score transformation and
to compute p-values using t-tests with pooled variance. If all measurements of a protein were
missing in one of the conditions, a pseudo fold change was computed replacing the missing
group average by the mean of 10% smallest protein intensities in that condition.

For IP experiments in tobacco followed by MS analysis, anti-HA IP on tobacco seedlings using
anti-HA beads (Anti-HA high affinity, Roche) was conducted as for immunoblot detection
(described above) without RNase treatment. After the final wash, the beads were re-suspended
in 45 µl digestion buffer (triethylammonium bicarbonate, pH 8.2), reduced with 5 mM tris(2-
carboxyethyl)phosphine and alkylated with 15 mM chloroacetamide. Proteins were on-bead
digested for 16 h at 37°C using 5 µl of sequencing-grade trypsin (100 ng/µl in 10 mM HCl,
Promega). The supernatants were transferred into new tubes, the beads washed with 150 µl
trifluoroacetic acid (TFA) buffer (0.1% TFA, 50% acetonitrile) and combined with the first
supernatant. After drying, the samples were re-solubilized in 3% (v/v) acetonitrile, 0.1% (v/v)
formic acid and analyzed on the Orbitrap Exploris 480 mass spectrometer described above.
Peptides were separated at a flow rate of 300 nl min-1 by the following gradient: initial hold at
5% solvent B (0.1% formic acid in acetonitrile) / 95% solvent A (0.1% formic acid in water) for 3
min, followed by a gradient to 22% B / 65% A in 43 min, and to 32% B / 68% A in 5 min, a
cleaning step at 95% B for 10 min and re-equilibration at 5% B / 95% A for 10 min. MS settings
were as follows: data-dependent mode (DDA) with a maximum cycle time of 3 s, acquisition of
full-scan MS spectra (350−1200 m/z) at a resolution of 120,000 at 200 m/z after accumulation to
a target value of 3,000,000 or for a maximum injection time of 50 ms, selection of precursors
with an intensity > 5,000 for MS/MS. Ions were isolated with a 1.2 m/z isolation window,
fragmented by higher-energy collisional dissociation using a normalized collision energy of 30%,
spectra acquired at 30,000 resolution, with maximum injection time set to auto. Singly,
unassigned and charge states higher than six were rejected. Precursor masses previously
selected for MS/MS measurement were excluded from further selection for 20 s, and the
exclusion window was set at 10 ppm. The samples were acquired using internal lock mass
calibration on m/z 371.1012 and 445.1200.

The mass spectrometry data were further processed using Philosopher84 followed by label-free
quantification based on precursor intensities using IonQuant85. MS2 spectra were searched
against the N. tabacum UniProt reference proteome (UP000084051) concatenated to its
reversed decoyed database and supplemented with MKIP1 proteins annotated in N. sylvestris
[A0A1U7XZG9_NICSY, A0A1U7Y1B8_NICSY] and common contaminants by the MSFragger76
search engine (v3.4), allowing for one missed tryptic cleavage and fixed carbamidomethylation
of cysteine, variable methionine oxidation, and variable acetylation of the protein N-terminus
after methionine removal. Quantification was performed by IonQuant85 (v1.7.17), applying the
false-discovery-rate controlled match-between-runs algorithm. Fold changes and adjusted p-
values were calculated using the Amica webserver77 (v3.0.1) using the following analysis
options: LFQ intensities for quantification, filtering for at least 3 MS/MS counts and at least 2
unique peptides, filter for proteins detected in at least three replicates in at least one group, no
re-normalization, imputation method “normal” (with imputation downshift = 1.8 and width = 0.3),
and “limma” for differential expression analysis.

Sucrose gradient centrifugation
Approximately 60 g of leaves from 24-day-old NtMatK C+ tobacco or Col-0 wild-type
Arabidopsis plants were homogenized in 500 ml homogenization buffer (50 mM HEPES-KOH,
pH 7.8, 330 mM sorbitol, 2 mM EDTA, 1 mM MgCl₂, 50 mM sodium ascorbate, 0.25% [w/v]
BSA) using a blender. The homogenate was filtered through two layers of Miracloth
(Calbiochem) and centrifuged at 2,000 g for 5 min at 4°C to enrich intact chloroplasts. The pellet
was resuspended in 15 ml lysis buffer (50 mM HEPES-KOH, pH 7.8, 1 mM MgCl₂) and
incubated on ice for 1 h to lyse the chloroplasts, followed by three rounds of grinding with a
glass homogenizer. The homogenate was centrifuged at 100,000 g for 20 min at 4°C to
sediment the chloroplast membranes. Proteins in the resulting supernatant were concentrated
using an Amicon Ultra-4 (10 kDa molecular weight cut off) filter column by centrifugation at
4,000 g at 4°C until the volume was reduced to 3 ml. For RNase treatment of Arabidopsis
extracts, RNase A (100 µg ml-1) and RNase T1 (250 U ml-1) (RNase A/T1 mix; Thermo Fisher)
were mixed with the stromal extracts and incubated on ice for 20 min.

Gradients of 10-30% (w/v) sucrose were prepared as previously described65 with some
modifications. Briefly, 30%, 25%, 20%, 15%, and 10% (all w/v) sucrose solutions were prepared
in 30 mM HEPES pH 8.0, 10 mM MgCl2, 60 mM KCl, and 5 mM DTT. Sequential 2-ml layers of
each sucrose solution were carefully added on top of one another in 13.2 ml Thinwall Ultra-
Clear tubes (Beckman Coulter). Each layer was frozen at –80°C before the next one was
added. The completed gradients were thawed for 16 h at 4°C prior to use. Fresh stromal extract
(500 µl, containing 2 mg protein) was carefully layered on top of the sucrose gradient. Gradients
were centrifuged in an SW41 Ti rotor (Beckman Coulter) at 274,000 g (40,000 rpm) for 16 h at
4°C, set to maximum acceleration and deceleration. Eleven 875 µl fractions were sequentially
collected from the top to the bottom of the gradient. The final (12th) fraction was pipetted up and
down to resuspend any pellet.

For immunoblotting, proteins in 100-µl aliquots of each sample fraction were subjected to
methanol-chloroform precipitation as described previously86. The precipitated proteins were
dissolved in 50 µl Laemmli buffer and boiled at 95 °C for 5 min, and 10 µl was loaded for each
blot. For anti-HA IP, 100 µl of each sample fraction were incubated with 5 µl anti-HA magnetic
beads (Pierce) at 4°C for 1 h with rotation, followed by three washes with chloroplast lysis
buffer. The washed beads were resuspended in 20 µl Laemmli buffer, boiled at 95 °C for 5 min,
and 10 µl of the eluted proteins was used for loaded for blotting.

Protein standards of the high-molecular weight gel filtration calibration kit (Cytiva) were
prepared and concentrated according to the manufacturer’s instructions. They were fractionated
in parallel with the chloroplast extracts. Standard protein migration was assessed by Coomassie
staining of methanol-chloroform precipitated fractions on SDS-PAGE gels.

Size exclusion chromatography and MS/MS analysis
Concentrated stromal extracts from NtMatK C+ tobacco were prepared as described for sucrose
gradient centrifugation. Stromal extract (500 µl, containing 2 mg protein) was loaded onto a
Superose 6 Increase 10/300 column on an ÄKTA Pure 25 system. Elution was performed with
chloroplast lysis buffer (50 mM HEPES-KOH, pH 7.8, 1 mM MgCl₂) at a flow rate of 0.25 ml min-
1 and 1 ml subfractions were collected. Subfractions 1–6 and 7–11 were pooled as sample
fractions 1 and 2, respectively, and reduced to 1 ml each using Amicon Ultra-4 (10 kDa
molecular weight cut off) columns by centrifugation at 4,000 g at 4°C. Other 1-ml subfractions
(numbered 3 to 14) were kept individually. Protein standards of the high-molecular weight gel
filtration calibration kit (Cytiva) were prepared and concentrated according to the manufacturer’s
instructions and fractionated and eluted as the chloroplast extracts. Anti-HA IP and precipitation
of 100-µl aliquots for immunoblotting were conducted as described for sucrose gradients.

For MS/MS analysis, 300 µl of selected fractions were precipitated in 80% (v/v) acetone. The
protein pellet was resuspended in 45 µl 10 mM Tris (pH 8.0), 2 mM CaCl2. Proteins were
reduced with 5 mM tris(2-carboxyethyl)phosphine and alkylated with 15 mM chloroacetamide at
30°C for 30 min, then digested using 250 ng trypsin (Sequencing Grade, from Promega) for 16
h at 37°C. After drying the peptides, they were re-solubilized in 20 µl 3% (v/v) acetonitrile, 0.1%
(v/v) formic acid and spiked with iRT peptides (Biognosys). Mass spectrometry analysis was
performed using the Orbitrap Exploris 480 mass spectrometer, columns and eluents A and B as
described above. For each sample, 200 ng of peptides were separated at 50°C at a flow rate of
300 nl min-1 using the following gradient: initial hold at 5% B for 3 min, followed by a gradient
from 5 to 22% B in 40 min, then 22 to 32% B in 10 min, a cleaning step at 95% B for 10 min and
re-equilibration at 5% B / 95% A for 10 min (all percentages v/v). The mass spectrometer was
operated in DIA mode. DIA scans covered a range from 400 to 960 m/z in windows of 8 m/z.
The resolution of the DIA windows was set to 30,000, with a normalized AGC target value of
1000%, the maximum injection time set to auto and a fixed normalized collision energy (NCE) of
30%. Each instrument cycle was completed by a full MS scan monitoring 396 to 1000 m/z at a
resolution of 60,000.

The DIA-MS raw files were further processed using MSConvert and analyzed using DIA-NN
implemented in FragPipe as described in section “Proteome analysis of β-estradiol treated
plants” but searching against the N. tabacum UniProt reference proteome (UP000084051)
concatenated to its reversed decoyed database and supplemented with MKIP1 proteins
annotated in N. sylvestris [A0A1U7XZG9_NICSY, A0A1U7Y1B8_NICSY] and common
contaminants. The two samples were analyzed together for spectral library built and protein
quantification, then analyzed separately to obtain the numbers of peptides identified in the
individual fractions. Only proteins identified with at least two peptides were kept for downstream
analysis.

RNA co-immunoprecipitation and sequencing
Soluble cell components were extracted by homogenizing 10 g of seedlings per replicate in a
pre-cooled blender using 30 ml of ice-cold extraction buffer (50 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 1 mM DTT, 1% (v/v) Triton X-100, 1 mM EDTA, 4 mM MgCl2, 1x protease inhibitor
cocktail [cOmplete EDTA-free, Roche], 40 U ml-1 RNase inhibitor [RiboLock RNase Inhibitor,
Thermo Fisher Scientific]). The homogenates were filtered through two layers of Miracloth
(Milipore) and cleared by two sequential centrifugation rounds at 4,000 g for 10 min at 4°C. Per
replicate, 20 ml of the supernatant were incubated with 150 µl pre-equilibrated magnetic
agarose anti-YFP beads (GFP-Trap, ChromoTek) for 2 h at 4°C with rotation, washed three
times with ice-cold wash buffer 1 (same as extraction buffer, but replacing Triton X-100 with
0.5% [v/v] NP-40), followed by three washes with ice-cold wash buffer 2 (50 mM Tris–HCl, pH
8.0, 150 mM NaCl). In the last wash, 10% (v/v) of the resuspended beads were collected in a
separate tube and used for immunoblot analysis, while the residual beads were transferred to a
new microcentrifuge tube and were processed for RNA extraction. For RNA extraction, 500 µl of
input soluble protein extract or IP-processed beads were brought to a total volume of 1 ml with
DEPC-treated water, mixed with 1 ml phenol:chloroform:isoamyl alcohol (Roth) and subjected to
phenol-chloroform RNA extraction as described previously87. Two µg input RNA was treated
with TURBO DNase (Thermo Fisher Scientific) in a 50 µl reaction according to the
manufacturer’s instructions. From this, 600 ng purified RNA was subjected to rRNA depletion
treatment as previously described88. The RNA was eluted in 18 µl of RNase-free water and
supplemented with 2 µl RNA fragmentation buffer (400mM Tris-acetate pH 8.3, 1 M potassium
acetate, 300 mM magnesium acetate), followed by incubation at 94°C for 5 min. Both pellet and
input RNA were treated with T4 polynucleotide kinase (PNK; Thermo Fisher Scientific) as
previously described88.

Library preparation for RNA sequencing was performed using the NEXTflex small RNA-seq kit
v3 (Perkin Elmer) according to the manufacturer’s instructions. The resulting cDNA was
amplified with a barcode incorporated in the primer and purified according to the instructions of
the NEXTflex kit. Libraries were pooled for single-end 100-bp sequencing in a NovaSeq 6000
machine. Sequencing reads were adapter-trimmed and filtered for size between 18 and 60 nt
using Cutadapt. The 8-nt UMI sequence in the RNA adapter was extracted, and reads were
deduplicated after mapping using umi_tools. Mapping was performed using STAR with the
Arabidopsis TAIR10 chloroplast genome (accession NC_000932.1) as reference, omitting the
second inverted repeat and using the following parameters: outFilterMismatchNoverLmax: 0.1;
alignIntronMin: 500; alignIntronMax: 1200; outSAMmultNmax: 1. Differentially abundant RNA
regions and the region boundaries were identified using DiffSegR89. Intron domains were
annotated according to the N. tabacum domain annotation in the CRW2 database90.

Total plant RNA extraction and northern blotting
Total plant RNA was extracted using GENEzol (Geneaid Biotech) according to the manufacturer
instructions with minor modifications. For visual observation of rRNAs, 10 µg of total RNA was
loaded onto a 1.5% agarose-formaldehyde gel (20 mM MOPS, pH 7, 8 mM sodium acetate,
1mM EDTA, 1.5% agarose, 6% formaldehyde) and separated by size by running the gel with
running buffer (20 mM MOPS, pH 7, 8 mM sodium acetate, 1mM EDTA, 3.7% formaldehyde) at
110 V for 3 h and stained with ethidium bromide. Quantification of rRNAs was conducted using
RNA ScreenTape by a TapeStation device (Agilent).

For RT-qPCR, first-strand cDNA synthesis was performed using the RevertAid First Strand
cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer instructions using
a 1:1 mixture of random hexamer primers and oligo(dT)18 primers for cDNA synthesis. After 10-
fold dilution, 1 µl cDNA was used in a 10 µl reaction of HOT FIREPol EvaGreen qPCR mix
(Solis BioDyne). qPCR reactions were performed using the LightCycler 480 II system (Roche).
For quantification of relative abundances of spliced and unspliced transcripts, abundances were
normalized to the AtRCE1 (AT4G36800) housekeeping gene91 before normalizing to the mean
of WT treated with β-estradiol. Primers are listed in Supplementary Data 1.

For northern blotting of intron/exon RNA, 10 µg of total RNA was resolved by a 1.5% agarose-
formaldehyde gel and transferred to HyBond-NX Nylon membrane (GE Healthcare) by capillary
transfer. The transferred RNA was then cross-linked to the membrane by two rounds of
ultraviolet light irradiation (1.2 kJ each). Radiolabelled probes against target transcripts were
generated using a Klenow-based labelling kit (Prime-a-Gene, Promega) using synthesized DNA
primers as template in the presence of [α-32P] dCTP. Probe hybridization, membrane washing,
and membrane exposure were conducted as previously described92, except that the
hybridization and subsequent washing steps were carried out at 60 °C. The primers used to
amplify the probes for analyzing intron splicing are listed in Supplementary Data 1. Northern
blots for assessing rRNA processing were conducted in the same way, except that 500 ng of
total RNA was resolved and transferred to 0.45 µm neutral nylon membranes (GVS North
America) by electrotransfer. Probes for analyzing plastidial rRNAs were described previously93.

Generation of yeast strains
Yeast strains, and constructs and primers used for creating them are detailed in
Supplementary Data 1. Constructs were made following the modular cloning toolkit for yeast94.
For AtMKIP1, the CDS of splice form 2 was used in all experiments. Plasmids were validated as
described for plant plasmids. Yeast transformation, transformant selection via hygromycin
resistance or uracil prototrophy, strain purification and confirmation of correct genomic
integration by PCR were conducted as previously described34.

For expression of YFP-tagged orthologous BEs, the CDS of BEs (without their cTPs, unless
cTP prediction was weak; Supplementary Data 1) were cloned as part 3a into pYTK001 entry
vector94 via BsmBI. They were then individually assembled with the other parts (the strong
constitutive CPW2 promoter, a C-terminal eYFP tag, and the CYC1 terminator) via BsaI into the
pre-assembled yeast integrative vector pBP296 (ref. 34), which targets the intergenic XII-5 yeast
locus95. The resulting vectors containing single transcription unit (TU) for BE expression were
transformed into the haploid CEN.PK113-11C yeast strain “578”34 (MATa MAL2-8C SUC2 his3Δ
malx2 glc3Δ gsy2Δ glg1Δ glg2Δ gph1Δ gsy1::pGAL1-EcglgC-TM-HA-tCYC1 bar1Δ XII-
2::pCWP2-mCherry-tUPT7:KanR gdb1::pUBI4-AtSS1-tRPL3).

For IP experiments in yeast, final yeast integrative vectors were targeted to the intergenic yeast
loci95 XII-5 (for testing homo-oligomerization of AtMatK) or XII-2 (all others tests)(Mikkelsen et al.,
2012). We therefore used either single TU constructs (for control strains expressing only a single
test protein) or multi TU constructs (for pair-wise interaction tests; Supplementary Data 1).
The AtMatK CDS (codon optimized for Arabidopsis and containing the edit frequently observed in
AtMatK RNA96, resulting in H236Y conversion) was first cloned as part 3a into pYTK001 via
BsmBI (pBP643), then assembled with parts for the promoter, a C-terminal tag and a terminator
via BsaI into the single-TU yeast vectors pBP662 (for the PGAL1:AtMatK-3xHA:TCYC1) or pBP773
(for PGAL1:AtMatK-FLAG:TCYC1). For expression of AtMKIP1-YFP and AtBE3-YFP without their
cTPs, the same TUs as for orthologous BE expression (PCWP2:AtMKIP1-YFP:TCYC1 from
pBP595; PCWP2:AtBE3-YFP:TCYC1 from pBP596) were used but targeting them to locus XII-2. An
additional single TU vector pBP774 was produced in an equivalent manner for expressing on
PCWP2:AtMKIP1-3xHA:TCYC1. For expression of AtMKIP1 fragments, the corresponding regions
(AA 120-333 for CBM-MSR; AA 178-333 for MSR; numbers based on the full-length AtMKIP1
protein including its cTP) were cloned as part 3a into pYTK001, then assembled into single-TU
vectors as the native AtMKIP1 protein. The ΔMSR -version of AtMKIP1 was created by
amplifying the AtMKIP1(ΔMSR) fragment from the vector AtMKIP1(ΔMSR)_pUBC-YFP and
assembling it into HindIII pre-digested pBP595 using the NEBuilder HiFi DNA Assembly kit
(New England Biolabs). N-terminal (AA 1-381) and C-terminal (AA 357-526) AtMatK segments
were created by deleting the other MatK regions in pBP662 by DNA assembly as above.

For pair-wise interaction tests, multi-TU integrative vectors were fused via BsmBI using the
preassembled integrative vectors pBP492 or pBP249, single-TU vectors and primer-based
spacers occupying empty positions. Final single- and multi-TU vectors were NotI digested and
transformed into the haploid CEN.PK113-11C strains “344” 34 (MATa MAL2-8C SUC2 his3Δ
glc3Δ gsy2Δ glg1Δ glg2Δ), or close descendants (Supplementary Data 1).

Light microscopy and branching enzyme activity gels in yeast
For light microscopy of iodine stained yeast cells, yeasts were inoculated in complex medium
with 2% (w/v) glucose (YPD) until saturation, diluted in complex medium with 2% (w/v)
galactose (YPGal, to induce expression of PGAL1-driven ADPglucose pyrophosphorylase) to an
OD of ~0.3 in Erlenmeyer flasks. After shaking for 5.75 h at 30°C and 260 rpm, cells were
harvested by centrifugation, washed with water, flash-frozen in liquid N2 and stored at -80°C.
Cells were thawn on ice, stained by mixing with Lugol’s solution in a 1:1 ratio, and immediately
imaged using an Axio Imager.Z2 light microscope equipped with a 100X oil-immersion objective
(Zeiss).

For protein analysis, yeasts were grown in the same way, but the main cultures were harvested
already after 3 h shaking in YPGal. Total and soluble protein extracts for immunoblotting were
prepared as previously described34. Immunoblotting was conducted as described above for
plant proteins. Native protein extraction and native PAGE monitoring BE activities (zymograms)
were conducted as previously described25.

Immunoprecipitation experiments in yeast
Yeasts were inoculated in YDP, grown for ~6h at 30°C and 260 rpm, then inoculated to an OD
of ~0.015 in Erlenmeyer flasks and shaken for 16 h at 30°C and 260 rpm. Cells were harvested
by centrifugation and once washed in water. Fresh cell pellets were resuspended in 3.3 volumes
of native protein extraction buffer (0.1 M MOPS, pH 7.75, 1% [v/v] Triton X-100, 1 mM EDTA, 4
mM MgCl2, protease inhibitor [Complete EDTA-free; Roche]) and 4.3 volumes of glass beads
(acid-washed, 425–600 mm diameter) and homogenized by vortexing for 30 min at 4°C. The
homogenate was transferred without glass beads to a fresh tube and centrifuged at 16,000 g for
5 min at 4°C. Equal amounts of soluble proteins present in the supernatant were mixed with pre-
blocked and/or washed magnetic beads. In case of anti-HA beads (Anti-HA Magnetic Beads,
Pierce), beads had been pre-blocked by incubation for 30 min at 20°C in TBS-T (Tris-buffered
saline, 0.1% [v/v] Tween-20) supplemented with 5% (w/v) bovine serum albumin (BSA), then
washed with TBS-T and resuspended in native protein extraction buffer. Magnetic agarose anti-
YFP beads (GFP-Trap, ChromoTek) had been prepared by washing them once in wash buffer
(50 mM Tris-HCl, pH 8, 0.15 M NaCl, 1 mM DTT, 1% [v/v] Triton X-100) and resuspending them
in native protein extraction buffer. The protein-bead mixture was incubated on a rotating wheel
at 4°C for 1 h, then washed six times with wash buffer (for anti-YFP beads) or TBS-T (for anti-
HA beads), using a magnetic stand for bead recovery. In the last wash step, beads were
transferred to a fresh tube to remove proteins adhering to the plastic walls. Proteins bound to
the beads were eluted by boiling in SDS-PAGE loading buffer as described for Arabidopsis IP
above. Input samples are aliquots of soluble proteins not subjected to immunoprecipitation.
Replicates derive from individual main cultures.

Sequence analyses and protein structure prediction
Starch-branching enzyme protein sequences were identified by BLASTp using AtMKIP1, AtBE2,
AtBE3, and rice BEI (a class I BE) as query in multiple genome and transcriptome databases.
Sequences were manually curated to remove duplicates and sequences shorter than 200 amino
acids very likely constituting fragments. Sequences were aligned by MAFFT (v7) with the E-INS-
I algorithm. Phylogenetic analyses were conducted in PhyloBayes MPI (v1.8c) using the “CAT +
GTR” substitution model. Two independent runs were performed, each cycling until the sampled
tree had stabilized and reached satisfactory convergence (maxdiff <0.3). A convergent tree was
calculated from the two runs and the posterior probabilities computed, discarding the first 500
cycles (< 5% of total cycles) of each run as burn-in. The unrooted phylogenetic tree was plotted
using Interactive Tree of life (v6).

For protein conservation analysis, protein sequences of each BE class (defined by the
phylogenetic tree) were aligned using MAFFT (v7)97 with the E-INS-I algorithm. Motif logos of
regions of interest were constructed using WebLogo 398. BE domains were predicted by
SMART99, Interpro100 and a locally installed version of ChloroP101. Disordered regions were
predicted by AIUPred102. The MSR was inferred from the multiple sequence alignment. The
conserved regions in the central catalytic domain were defined as previously33. RT motifs and
the X domain of AtMatK base on the alignment to the NtMatK domains annotated previously17,
except that the X domain was slightly elongated (AA 382-495 instead of 382-483) to fully include
an α-helix predicted by AlphaFold3. Protein structures without their predicted cTP (if any) were
predicted by AlphaFold2 (for monomeric proteins; 103) or AlphaFold3 (for protein complexes; 104).
Interfaces in the four-subunit complex were detected by AlphaBridge105. Molecular graphics and
analyses were performed with UCSF ChimeraX (v1.10)106. The predicted binding sites are
contacts between AtMatK and AtMKIP1 within 4 Å distance with a maximum PAE value of 5 Å.
Only binding sites containing at least 3 contacts are shown, with a maximum gap of 2 AA
between contacts allowed.

Accession numbers
The Arabidopsis Genome Initiative gene codes associated with this study are: AT3G20440
(AtMKIP1/AtBE1), ATCG00040 (AtMatK), AT1G07320 (AtuL4c), AT5G16715 (AtValRS2),
AT3G14900 (AtEMB3120), AT2G36390 (AtBE3). Accession numbers for BE orthologs
expressed in yeast are given in Supplementary Data 1.

Statistical analyses
The data shown in Figs. 8f, 9c-e and Supplementary Fig. 15 were first evaluated for normal
distribution using the Shapiro-Wilk Test and tested for equal variance using Bartlett's test. Since
the data did not meet the assumptions of normal distribution and equal variance, the non-
parametric Kruskal-Wallis test was used for determining statistically significant differences
between the groups, followed by a two-sided Dunn's post hoc multiple comparisons test and
Bonferroni p value adjustment to identify the specific groups that are significantly different.
Statistical analyses of the mass spectrometry and RNA sequencing data is described in the
corresponding sections.

Data availability
All data needed to evaluate the conclusions in this paper are present in the paper and/or its
supplementary materials. The proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE107 partner repository with the identifiers PXD060108 (IP data of
AtMKIP1-YFP; reviewer token: 9E3plmRjS954), PXD060055 (IP data of NtMatK-HA; reviewer
token: 0ESPSHedZFDx), PXD069946 (MS/MS analysis of SEC fractions; reviewer token:
TDcsv4Ttg2TY), and PXD067382 (Proteomics after silencing of AtMKIP1 or AtuL4c; reviewer
token: ANBQ1UOYwrRs). The RNA sequencing data of RNAs bound to AtMKIP1-YFP will be
publicly available at NCBI GEO108 with the accession number GSE284378
(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE284378; reviewer token:
ghapyaqwrdmdnef). Microscopy images and structure predictions will be deposited, without
restrictions, at the ETH Zürich Data Archive. New genetic material (transgenic Arabidopsis and
yeast lines) and vectors will be made available to the scientific community upon request. Source
Data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Liang et al. describe the identification and molecular characterization of MKIP1 a chloroplast protein interacting with the plastid groupII intron splicing factor MatK. Unexpectedly, MKIP1 shares homologies with unrelated starch branching enzymes (BEs). However, the authors unambiguously demonstrate that MKIP1 has lost its BE activity and, instead, has acquired a new function in chloroplast RNA metabolism. Moreover, the comprehensive genetic and molecular work clearly shows that MKIP1 associates with intron RNAs and is involved in their splicing. The manuscript reads well and the findings provide new insights into the evolution and molecular players in organellar splicing. However, before publishing a few points might be considered.\nWhereas the genetic data convincingly reveals a role of MKIP1 during splicing, its precise function remains a bit diffuse. This is mainly due to the lack of experimental data on the detected MatK/MKIP1 RNP complex. What is the molecular size of this complex (e.g. in native PAGE)? What is the stoichiometry of the subunits? For instance, the blue coloring in the lower-right corner of the predicted aligned error (PAE) plot (Fig. 4a), corresponding to intermolecular regions of AtMKIP1, suggests potential homodimerization or higher-order oligomeric states for AtMKIP1. To investigate such alternative conformations, the application of AlphaFold3 would be helpful (Abramson et al., 2024). Additionally, AlphaFold3 could be utilized to predict protein–nucleic acid interactions, enabling a more detailed understanding of the recruitment order within this functional complex. Such predictions could significantly refine the proposed model illustrated in Fig. 8.\nIn line with this, does recombinant MKIP1 produced in yeast has RNA binding affinity on its own?\nMinor points:\n- Fig.1b: It is stated in the text (line 350) that AtMKIP1-YFP lacked a punctuate sub-organellar localization pattern typical for nucleoids. However, to this reviewer some chloroplasts in Fig. 1b do display a punctuate pattern. Maybe imaging can be improved.\n- Fig. 1e: In the Figure legend, it should be given what the difference between the two last images from true leaves is.", "reviewer_2": "review to Nat. Comm (NCOMMS-24-81917) “Maturase K forms a plastidial splicing complex with a neofunctionalized branching enzyme”\nPlastids typically harbor introns in various essential genes that must be removed post-transcriptionally to allow the functionality of the mature transcripts. The work of Yuanyuan Liang et al focuses on MKIP1, an essential gene encoding a plastid-localized homolog of starch-branching enzymes (BEs). The authors indicate that MKIP1 lacks BE activity and instead acquired features that enable it to facilitate gII’s processing in coordination with the intron-encoded MatK proteins. The two proteins can interact with one another independently of gII intron RNAs. The authors further indicate a role for MKIP1’s in RNA-binding and established embryo rescue or (estradiol-inducible) micro-RNA knockdown lines to investigate its roles in cp-RNA metabolism. The RNA profiles showed that MKIP1 mutant lines are indeed affected in gIIA’s splicing (excluding ClpP), which is similar to suggested roles of MatK protein (Zoschke et al 2020). Additionally, co-IPs indicated that AtMKIP1 is associated with multiple group IIA introns in vivo, again in a similar manner to those of MatK. Overall, this reviewer finds that the data is original and strengthens the study's impact on RNA metabolism in the organelles of plants. The findings of MKIP1 roles in plastid gII intron splicing and interaction with MatK are novel and convincing, although I find the suggested model of action less supported by the data and previous in vitro analyses of MatK. Likewise, the work, figures and the data organization are definitely of good quality. The clarity of the methodology and the overall quality of the data obviously make it a valuable contribution to the field. I have some comments to the authors that may improve the data and reading of the MS.\nSpecific comments regarding the MS data and text:\n1. The analysis of the mutants needs further support. i.e., as indicated Fig 1 relates to “hemi-complemented” (genetically-rescued) ABI3 lines, while the RNA and protein profiles relate to estradiol-inducible amiR lines. I find this confusing. more importantly, it would be more convincing to show that all the data, e.g., organellar ultrastructural morphologies, as well as the plastidial RNA and protein profiles are done for all the genetic lines (amiR and ABI3 lines)\n2. The effects of the mutations of RNA metabolism are convincing (excluding comment #1), but less so for the overall protein profiles. The authors indicate translation defects for a few plastid-encoded proteins. One would assume that altered processing of transcripts that correspond to tRNAs and ribosomal factors (Fig. 7 and Fig. S10) would generally affect organellar gene expression. Unclear whether this is indeed supported by the mutant’s protein data.\n3. The lower blot in Fig 7 is cut for the corresponding RbcL bands! it would also be more convincing to show the complete blots.\n4. Also, regarding comments above - how do the relatively mild effects seen in the RNA and protein data correlate with the strong defects in plant physiology?\n5. The background on group II introns is comprehensive but assumes prior knowledge. Adding a short/brief explanation of their function and relevance in plants could help readers that are unfamiliar with the topic.\n6. The transition to discussing MatK and its divergence from bacterial maturases is a bit abrupt. Consider smoother transitions.\n7. MKIP1 gene locus, and gene expression. Notably, the AT3G20440 locus at TAIR database has alternative splicing variants, one of which affects the N-terminal region (AT3G20440.3). Is there a reason that the authors focus on one of these for the genetic analysis AT3G20440.2?\n8. How the authors explain the association of MKIP1 with itself and with the other splicing variants (IP MS data in suppl. data)? do they all correspond to the same peptides? or rather co-IPed the two paralogous variants.\n9. Fig. 1 - Localization analysis. The confocal microscopy requires additional controls (mito’s , nuclear at cetera). Note that MKIP1 was identified in a nuclear MS/MS analysis (although may correspond to chloroplast contamination).\n10. Following their data, and in light of Fig 6c (MS results lines 292-294), the authors need to show the global effects of MKIP1 mutations on ribosome biogenesis effects (e.g., sucrose gradients, RNase protection assays of plastidial rRNAs) as well as on chloroplast translation (i.e., MS/MS).\n11. The model in Fig 8 is not supported, and might be wrong. Other than a few canonical MAT’s, the association of MatK with group IIA intron RNA targets is indeed currently unknown. However, MatK was previously shown to demonstrate some specificity in its association with its defined gII’s targets in vitro,this in the absent of additional protein cofactors (see Liere, K. and Link, G. (1995) RNA-binding activity of the Matk protein encoded by the chloroplast trnK intron from mustard (Sinapis-Alba L). Nucleic Acids Res, 23, 917-921.; Barthet, M.M., Pierpont, C.L. and Tavernier, E.K. (2020) Unraveling the role of the enigmatic MatK maturase in chloroplast group IIA intron excision. Plant Direct, 4, e00208. MatK protein contains part of the RT0 sequence motif (a region with importance for specific gII RNA binding), although most of this region (and other T elements) seem indeed missing from MatK.\nSome other minor remarks:\n1. The abstract is well-written, but some sentences are lengthy and could benefit from simplification for clarity.\n2. The authors may want to briefly explain at the first paragraph in the intro/abstract how At-MKIP1 was originally identified as a MatK interacting partner (co-IPs?)\n3. \"Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK.\" Could be rephrased (e.g., \"In Arabidopsis, MKIP1 co-precipitates with all known intron targets of MatK.\")\n4. The concluding statement about the N-terminus of MatK could be clearer if rephrased.\n5. The introduction is quite clear but occasionally verbose. For example: \"Chloroplast gene expression involves the splicing of ~20 introns, a process essential for plant viability.\" Consider to alter and to combine with the subsequent sentence for a smoother transition.\n6. The section on MatK's conserved X domain could use reorganization for better logical flow.\n7. Results line 177 - EMB3120 is annotated at TAIR as - SNOW-WHITE LEAF1, SWL1 (see REF of Yue Wang et al Crucial role of SWL1 in chloroplast biogenesis and development in Arabidopsis thaliana. Plant Cell Rep 43, 135 (2024). https://doi.org/10.1007/s00299-024-03210-1."}, "author_response": "Responses to the reviewers\nWe would like to thank both reviewers for their valuable suggestions, which we believe have\nhelped us improve the manuscript. We were able to address almost all concerns except in a\nfew cases where we encountered technical limitations. Additionally, we updated all multimer\npredictions from AlphaFold2 to AlphaFold3 to ensure consistency.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Liang et al. describe the identification and molecular characterization of\nMKIP1 a chloroplast protein interacting with the plastid groupII intron splicing factor MatK.\nUnexpectedly, MKIP1 shares homologies with unrelated starch branching enzymes (BEs).\nHowever, the authors unambiguously demonstrate that MKIP1 has lost its BE activity and,\ninstead, has acquired a new function in chloroplast RNA metabolism. Moreover, the\ncomprehensive genetic and molecular work clearly shows that MKIP1 associates with intron\nRNAs and is involved in their splicing. The manuscript reads well and the findings provide\nnew insights into the evolution and molecular players in organellar splicing. However, before\npublishing a few points might be considered.\nWhereas the genetic data convincingly reveals a role of MKIP1 during splicing, its precise\nfunction remains a bit diffuse. This is mainly due to the lack of experimental data on the\ndetected MatK/MKIP1 RNP complex. What is the molecular size of this complex (e.g. in\nnative PAGE)? What is the stoichiometry of the subunits? For instance, the blue coloring in\nthe lower-right corner of the predicted aligned error (PAE) plot (Fig. 4a), corresponding to\nintermolecular regions of AtMKIP1, suggests potential homodimerization or higher-order\noligomeric states for AtMKIP1. To investigate such alternative conformations, the application\nof AlphaFold3 would be helpful (Abramson et al., 2024).\nResponse: We thank the reviewer for the excellent recommendation to characterize the\nmolecular weight and composition of the MatK complex further and to implement more\nAlphaFold3 predictions. While we were unable to detect the complex subunits in native\nPAGE, we could analyse the migration of the complex in sucrose density gradients and its\nelution using size exclusion chromatography. For this, we used the tobacco NtMatK C+ line,\nwhich allowed us to detect MatK-HA by western blotting, as well as Arabidopsis. These data\nshow that NtMatK, NtMKIP1, NtValRS2 and NtEMB3120 co-migrate into high-molecular-\nweight fractions, suggesting that they together form a stable complex (new Fig. 4 and new\nSupplementary Fig. 9). This is consistent with AlphaFold3 prediction of a four-subunit\ncomplex with a 1:1:1:1 stoichiometry of AtMatK, AtMKIP1, AtValRS2 and AtEMB3120 with\nrelatively high confidence (ipTM = 0.7; new Fig. 5).\nWe refrain from estimating an exact molecular weight of the complex from these methods\nsince these estimations can be biased for non-globular protein complexes. However, we\ntested for possible self-association of AtMKIP1, AtMatK, AtValRS2 and AtEMB3120 after\nexpression in tobacco or in yeast (new Supplementary Fig. 10). None of the proteins\nshowed significant self-association. This is again consistent with the AlphaFold3 predictions,\nwhich did not support homo-oligomerization for any subunit. AlphaFold3 predictions also did\nnot support the association of a second molecule of either subunit in the presence of the\nother complex components (new Supplementary Fig. 10). Collectively, these data suggest\nthat the complex exists in the form of a tetramer with one subunit of each MatK, MKIP1,\nValRS2 and EMB3120. The observed molecular weight ranges of the complex by size-\nexclusion chromatography and sucrose density gradient centrifugation would be certainly\nplausible for a tetramer (~340 kDa, not counting any associated RNA fragments). While more\ndetails on the composition and structure would be very interesting, we feel that they are\nbetter suited for follow-up work.\nWe would like to clarify that the PAE plot of the AtMatK-AtMKIP1 heterodimer (now in Fig.\n6d) only indicates the confidence in the relative placement of the AtMatK and AtMKIP1\nresidues, and does not allow any conclusions to be drawn about the potential homo-\noligomerisation of either subunit.\nAlphaFold3 could be utilized to predict protein–nucleic acid interactions, enabling a more\ndetailed understanding of the recruitment order within this functional complex. Such\npredictions could significantly refine the proposed model illustrated in Fig. 8.\nResponse: Although AlphaFold3 is better than previous versions at predicting RNA folding,\nthese predictions remain difficult, particularly for large RNAs such as group II introns.\nUnfortunately, the plastidial group II intron structures predicted by AlphaFold3 did not\nresemble the experimentally determined structures of related bacterial group II introns.\nAlphaFold3 was also unable to predict smaller intron fragments of around 50 nucleotides with\nreasonable confidence. Given the high likelihood that RNA structures are not being predicted\ncorrectly, we lean away from predicting their interaction with the MatK complex and feel that\nfuture work should aim to experimentally determine the structure of the RNP complex. In this\nlight, we agree that the model originally presented in Fig. 8 is not sufficiently supported and\nhave thus removed it. We have also added a statement in the discussion regarding the\ndifficulties in modelling group IIA introns and their association with the complex.\nIn line with this, does recombinant MKIP1 produced in yeast has RNA binding affinity on its\nown?\nResponse: We thank the reviewer for this useful suggestion. We spent a considerable\namount of time obtaining soluble proteins of good purity and testing different conditions for\nRNA binding. Ultimately, we managed to express and purify AtMKIP1 (and AtBE3 as a\ncontrol) fused to an N-terminal 6x-His-SUMO tag in E. coli, albeit yielding relatively low\nconcentrations. We tested each domain of the Arabidopsis trnK intron for RNA binding using\nelectrophoretic mobility shift assays. We repeatedly observed the association of the tagged\nAtMKIP1 with the complete DVI, but not with any of the other tested RNA regions. No\nsignificant binding of AtBE3 to DVI was observed. However, as the extent of AtMKIP1\nbinding varied between experiments and binding saturation could not be observed, we could\nnot estimate affinities. We believe that establishing AtMKIP1's RNA binding would require\nfurther optimization and the combination of multiple techniques (ideally incorporating\nstructural information of the RNP complex – see above) to achieve conclusive, robust, and\nbiologically meaningful results. This follow-up work is certainly very interesting but is beyond\nthe scope of this manuscript.\nMinor points:\n- Fig.1b: It is stated in the text (line 350) that AtMKIP1-YFP lacked a punctuate sub-\norganellar localization pattern typical for nucleoids. However, to this reviewer some\nchloroplasts in Fig. 1b do display a punctuate pattern. Maybe imaging can be improved.\nResponse: We agree that AtMKIP1-YFP appeared somewhat inhomogeneously distributed\nwithin the chloroplasts. This may reflect an uneven distribution of AtMKIP1-YFP within the\nstroma due to the presence of thylakoid membranes, which differs from the distinct punctae\ntypically observed for nucleoids (see, for example, Fig. 1A from Powikrowska et al., 2014.\nDynamic composition, shaping and organisation of plastid nucleoids. Frontiers in Plant\nScience, https://doi.org/10.3389/fpls.2014.00424). To clarify, we have replaced this image\nwith more detailed micrographs. In response to a suggestion from Reviewer #2, these also\nshow the locations of the mitochondria and nuclei (Fig. 1b and new Supplementary Fig. 3).\nThe new data indicate that, under the tested conditions, AtMKIP1 localises exclusively to the\nplastids, where it appears to be distributed fairly evenly within the stroma.\n- Fig. 1e: In the Figure legend, it should be given what the difference between the two last\nimages from true leaves is.\nResponse: We have now indicated some of the structures observed within the plastids with\narrows and added the following statement to the legend: “In the true leaves from rescued\nmkip1-1-/- plants, plastids typically contained collapsed thylakoids (red arrow) and vesicular\nstructures (yellow arrow). Occasionally, larger thylakoid stacks were observed (outer right\nimage).”\nReviewer #2 (Remarks to the Author):\nPlastids typically harbor introns in various essential genes that must be removed post-\ntranscriptionally to allow the functionality of the mature transcripts. The work of Yuanyuan\nLiang et al focuses on MKIP1, an essential gene encoding a plastid-localized homolog of\nstarch-branching enzymes (BEs). The authors indicate that MKIP1 lacks BE activity and\ninstead acquired features that enable it to facilitate gII’s processing in coordination with the\nintron-encoded MatK proteins. The two proteins can interact with one another independently\nof gII intron RNAs. The authors further indicate a role for MKIP1’s in RNA-binding and\nestablished embryo rescue or (estradiol-inducible) micro-RNA knockdown lines to investigate\nits roles in cp-RNA metabolism. The RNA profiles showed that MKIP1 mutant lines are\nindeed affected in gIIA’s splicing (excluding ClpP), which is similar to suggested roles of\nMatK protein (Zoschke et al 2020). Additionally, co-IPs indicated that AtMKIP1 is associated\nwith multiple group IIA introns in vivo, again in a similar manner to those of MatK. Overall,\nthis reviewer finds that the data is original and strengthens the study's impact on RNA\nmetabolism in the organelles of plants. The findings of MKIP1 roles in plastid gII intron\nsplicing and interaction with MatK are novel and convincing, although I find the suggested\nmodel of action less supported by the data and previous in vitro analyses of MatK. Likewise,\nthe work, figures and the data organization are definitely of good quality. The clarity of the\nmethodology and the overall quality of the data obviously make it a valuable contribution to\nthe field. I have some comments to the authors that may improve the data and reading of the\nMS.\nSpecific comments regarding the MS data and text:\n1. The analysis of the mutants needs further support. i.e., as indicated Fig 1 relates to “\nhemi-complemented” (genetically-rescued) ABI3 lines, while the RNA and protein profiles\nrelate to estradiol-inducible amiR lines. I find this confusing. more importantly, it would be\nmore convincing to show that all the data, e.g., organellar ultrastructural morphologies,\nas well as the plastidial RNA and protein profiles are done for all the genetic lines (amiR and\nABI3 lines)\nResponse: We thank the reviewer for these useful suggestions. Unfortunately, there is very\nlittle tissue available from the seedling-lethal P :AtMKIP1-mCitrine+/+/mkip1-/- plants.\nABI3\nDespite pooling homozygous mutant seedlings from multiple plates, we were unable to\nrecover sufficient RNA to test for splicing deficiencies using northern blotting with multiple\nreplicates. Nevertheless, we analysed all non-tRNA introns using RT-qPCR and identified\nsignificant splicing defects in the MatK/MKIP1-associated group IIA introns (i.e. in intron 2 of\nrps12 and the introns in rpl2 and atpF). These splicing defects were approximately twice as\nstrong as those observed in the amiR-mkip1 lines. These data are consistent with the strong\nvisual phenotypes and with a role for AtMKIP1 in the splicing of these introns. However,\ngiven the seedling lethality of this line, we expect this tissue to exhibit pleiotropic defects,\nincluding severe impairment of plastidial translation. This makes identification of the primary\nmolecular defect difficult, particularly since we lack an appropriate control line with a primary\ndefect in plastidial translation. For these reasons, we did not include the limited splicing data\nwe have in the manuscript.\nNone of the problems described above apply to the amiR-mkip1 lines: material is sufficient,\nso no pooling is required, and the inducible system allowed us to harvest the tissue before\nstrong pleiotropic effects develop. In addition, we have an appropriate control line (amiR-\nuL4c) available to account for the mild defects in plastidial translation. In the revised version\nof the manuscript, we have included transmission electron micrographs of amiR-mkip1 from\nan earlier, comparable batch, which show that the plastidial ultrastructure is altered and\ncontains fewer thylakoid stacks (new Supplementary Fig. 12). In response to the reviewer's\nother concerns, we have expanded the molecular analyses to include a thorough\nassessment of proteome changes (Fig. 8 and new Supplementary Fig. 13), as well as\nrRNA analyses (new Supplementary Fig. 14). We have also added a statement at the\nbeginning of the silencing results section explaining the limitations of the P :AtMKIP1-\nABI3\nmCitrine+/+/mkip1-1-/- lines and the linkage between plastidial splicing and translation.\n2. The effects of the mutations of RNA metabolism are convincing (excluding comment #1),\nbut less so for the overall protein profiles. The authors indicate translation defects for a few\nplastid-encoded proteins. One would assume that altered processing of transcripts that\ncorrespond to tRNAs and ribosomal factors (Fig. 7 and Fig. S10) would generally affect\norganellar gene expression. Unclear whether this is indeed supported by the mutant’s\nprotein data.\nResponse: To analyse the impact of MKIP1 silencing on plastidial gene expression and\nabundance of plastome-encoded proteins at larger scale, we conducted total proteome\nanalysis by MS/MS in the three amiR lines and WT control. Indeed, most of the quantified\nplastidial ribosomal proteins of the large and small ribosomal subunits (new Supplementary\nFig. 13c) are significantly reduced in amiR-mkip1-1. The same applies to the quantified\nplastome-encoded proteins (Fig. 8b). Reduced levels of ribosomal and plastome-encoded\nproteins are also apparent in amiR-mkip1-2 but milder and mostly not statistically significant,\nconsistent with the generally milder reduction in MKIP1 and phenotypes of this line. These\ndata indicate that silencing of MKIP1 indeed has widespread negative effects on plastidial\ngene expression.\n3. The lower blot in Fig 7 is cut for the corresponding RbcL bands! it would also be more\nconvincing to show the complete blots.\nResponse: Unfortunately, the membrane had been physically cut just above the RbcL band\nin order to allow probing of one membrane against two proteins. However, we could enlarge\nthe visible area to (almost) fully show the RbcL bands in Fig. 8c. We note that RbcL is fully\nvisible in the blot above (which unfortunately had not been stained by coomassie/Revert 700)\nand RbcL has also been quantified by proteomics (Fig. 8d). All uncropped membranes are\ndisplayed in the Source Data file.\n4. Also, regarding comments above - how do the relatively mild effects seen in the RNA and\nprotein data correlate with the strong defects in plant physiology?\nResponse: In our view, the effect strengths are in-line with each other. The changes in\nspliced vs. unspliced RNA pools are quite large, as are the changes in chloroplast-localized\nproteins of the leaf. Chlorosis could be a direct result, or made worse by photoinhibition,\nresulting from an imbalance between light energy capture and its utilisation.\nIn leaves that had already matured before the induction of silencing, the level of MKIP1 also\ndecreases, but the stability of plastome-encoded proteins in the plastids is likely sufficient to\nmaintain photosynthesis and a green phenotype for the ~1-week period during which MKIP1\nlevels are reduced. Newly developing leaves, where the photosynthetic machinery must first\nbe established, are more affected, albeit with the expected delay. When we exposed amiR-\nMKIP1 plants to β-estradiol from the seed or seedling stage, their phenotypes were much\nmore severe, with albino leaves developing from the beginning. However, we did not further\nanalyse such tissues because of the likely pleiotropic effects (see response to comment no.\n1, above).\n5. The background on group II introns is comprehensive but assumes prior knowledge.\nAdding a short/brief explanation of their function and relevance in plants could help readers\nthat are unfamiliar with the topic.\nResponse: We expanded the description of the function and relevance of plastidial introns at\nthe beginning of the introduction.\n6. The transition to discussing MatK and its divergence from bacterial maturases is a bit\nabrupt. Consider smoother transitions.\nResponse: We have added a link to the previous paragraph to make the transition\nsmoother.\n7. MKIP1 gene locus, and gene expression. Notably, the AT3G20440 locus at TAIR\ndatabase has alternative splicing variants, one of which affects the N-terminal region\n(AT3G20440.3). Is there a reason that the authors focus on one of these for the genetic\nanalysis AT3G20440.2?\nResponse: In addition to sf2 (At3g20440.2), which we used in our work, there are two other\nsplice form (sf) variants (sf1 [At3g20440.1] and sf3 [At3g20440.3]) annotated on TAIR. The\ncorresponding proteins differ in their middle-to-C-terminal regions (note that At3g20440 is on\nthe minus strand and is therefore read from right to left in genome browsers): Compared to\nthe protein from sf2, the protein from sf3 has a total of seven amino acids inserted and nine\ndeleted between amino acids 365 and 881. The protein from sf1 has a deletion of 30 amino\nacids after lysine 423. While the mapping coverage of RNA sequencing data fully supports\nthe expression of sf2, it does not support expression of sf1 or sf3 mRNA in diverse tissues,\nincluding leaves and roots (see the 'RNA-seq based evidence' section of this locus at\nhttps://jbrowse.arabidopsis.org). In addition, no peptides specific to proteins from sf1 or sf3\nhave been reported in the proteomics database\n(https://www.proteomicsdb.org/proteomicsdb/#protein/proteinDetails/248572/peptides).\nWe therefore conclude that sf2 is the predominant, if not the sole, expressed version of\nMKIP1, which is consistent with its ability to complement the MKIP1 mutant phenotype. We\nhave added an explanation as to why sf2 was selected to the Methods section ‘Cloning of\nplant constructs’.\n8. How the authors explain the association of MKIP1 with itself and with the other splicing\nvariants (IP MS data in suppl. data)? do they all correspond to the same peptides? or rather\nco-IPed the two paralogous variants.\nResponse: In the IP-MS experiments conducted in Arabidopsis, all MKIP1 peptides detected\nmatch to its sf2, i.e. the form that was expressed with a YFP tag and used as bait. Most MKIP1\npeptides additionally match to sf1 and/or 3; as described above, they are highly similar to sf2.\nHowever, we did not detect any peptides specific to sf1 or sf3. The data thus do not provide\nevidence for self-association of MKIP1. In relation to a comment of reviewer #1, we also tested\nself-association of Arabidopsis MKIP1-YFP with MKIP1-FLAG in IP experiments after transient\nexpression in tobacco and could not observe homo-oligomerization (new Supplementary Fig.\n10).\n9. Fig. 1 - Localization analysis. The confocal microscopy requires additional controls (mito’\ns , nuclear at cetera). Note that MKIP1 was identified in a nuclear MS/MS analysis (although\nmay correspond to chloroplast contamination).\nResponse: We replaced the previous micrograph with more detailed confocal micrographs\nof Arabidopsis epidermal leaf cells. Using staining by MitoTracker and DAPI, we now also\nvisualise mitochondria and nuclei, in addition to AtMKIP1-YFP and chlorophyll\nautofluorescence (Fig. 1b and new Supplemental Fig. 3). Our microscopy analyses indicate\nthat AtMKIP1 exclusively localises to the plastids.\n10. Following their data, and in light of Fig 6c (MS results lines 292-294), the authors need to\nshow the global effects of MKIP1 mutations on ribosome biogenesis effects (e.g., sucrose\ngradients, RNase protection assays of plastidial rRNAs) as well as on chloroplast translation\n(i.e., MS/MS).\nResponse: In our initial visualization of rRNAs by gel staining we had only observed\nunprocessed 23S rRNA resulting from incomplete hidden breaks within the large ribosomal\nsubunit (now in Fig. 8e) in AtMKIP1 silenced tissue, and such specific defects on single\nrRNA species had indeed been linked before to potential primary defects in assembly of the\nlarge ribosomal subunit (Reiter et al., 2020). To examine this further, we have now analysed\nall plastidial rRNAs in detail by northern blotting (new Supplementary Fig. 14). These blots\nshow that 1) both AtMKIP1 and AtuL4c silencing leads to aberrant processing of plastidial\nrRNAs, and 2) that rRNAs of both the small and large plastidial ribosomal subunits are\naffected. Importantly, this points towards a global plastidial rRNA processing defect, which is\na common secondary outcome of mutations affecting plastidial translation: very similar rRNA\nprocessing patters as upon AtMKIP1 silencing have been observed in Arabidopsis mutants\nof ribosomal proteins of the plastidial large or small ribosomal subunits (Tiller et al., 2012)\nand the maize ppr5 mutant (Beick et al., 2008), which has a primary defect in tRNA\nprocessing and thus secondarily in plastidial translation.\nAs described in the answers to comment 2, our total proteome analyses points towards a\ndefect in plastidial translation upon AtMKIP1 silencing. This in turn will affect ribosome\nassembly and, as multiple rRNA processing steps occur after rRNA incorporation into the\nribosome, also rRNA processing. However, we stress that these defects are most likely\nsecondary consequences of the group IIA intron splicing defects, and the associated\nreduction in the abundance of mature tRNAs and ribosomal proteins. The view that the\nalterations in rRNA processing and plastid translation upon MKIP1 silencing are indirect is\nconsistent with our observation that NtMKIP1 does not co-migrate with a component of the\nlarge ribosomal subunit (NtuL4c) in sucrose gradients / size exclusion experiments (new Fig.\n4 and Supplementary Fig. 9).\n11. The model in Fig 8 is not supported, and might be wrong. Other than a few canonical\nMAT’s, the association of MatK with group IIA intron RNA targets is indeed currently\nunknown. However, MatK was previously shown to demonstrate some specificity in its\nassociation with its defined gII’s targets in vitro, this in the absent of additional protein\ncofactors (see Liere, K. and Link, G. (1995) RNA-binding activity of the Matk protein encoded\nby the chloroplast trnK intron from mustard (Sinapis-Alba L). Nucleic Acids Res, 23, 917-\n921.; Barthet, M.M., Pierpont, C.L. and Tavernier, E.K. (2020) Unraveling the role of the\nenigmatic MatK maturase in chloroplast group IIA intron excision. Plant Direct, 4, e00208.\nMatK protein contains part of the RT0 sequence motif (a region with importance for specific\ngII RNA binding), although most of this region (and other T elements) seem indeed missing\nfrom MatK.\nResponse: As discussed in response to Reviewer 1, we recognize that that the model\noriginally presented in Fig. 8, is not yet sufficiently supported and have thus removed it.\nFurther structural studies will be undertaken to determine the mechanism by which MatK\nassociates with its different intron targets and how the other proteins we have identified\ncontribute to its function. We also now mention the earlier evidence that MatK by itself can\nstill bind to RNA to some extent (Barthet & Hilu, 2008; Liere & Link, 1995) in the discussion.\nIntron binding is likely conferred by the X domain of MatK, while the contribution of the N-\nterminal half of this process is unclear. Even though the N-terminus may still have a part of\nthe RT0 sequence and may contribute to RNA binding, conservation of the RT0 is restricted\nto few amino acids, and these are now predicted with high confidence to make close\ncontacts to MKIP1. Given the general sequence divergence of MatK’s N-terminal region and\nits observed and predicted engagement in protein-protein interactions, we find it plausible\nthat this region does not fullfil its original function anymore, which includes establishing the\nhigh-affinity contacts with intron RNA.\nSome other minor remarks:\n1. The abstract is well-written, but some sentences are lengthy and could benefit from\nsimplification for clarity.\nResponse: We have adapted the abstract accordingly.\n2. The authors may want to briefly explain at the first paragraph in the intro/abstract how At-\nMKIP1 was originally identified as a MatK interacting partner (co-IPs?)\nResponse: We have adapted the abstract accordingly.\n3. \"Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK.\" Could be\nrephrased (e.g., \"In Arabidopsis, MKIP1 co-precipitates with all known intron targets of\nMatK.\")\nResponse: We have adapted the abstract accordingly.\n4. The concluding statement about the N-terminus of MatK could be clearer if rephrased.\nResponse: We have changed the end of the abstract accordingly.\n5. The introduction is quite clear but occasionally verbose. For example: \"Chloroplast gene\nexpression involves the splicing of ~20 introns, a process essential for plant viability.\"\nConsider to alter and to combine with the subsequent sentence for a smoother transition.\nResponse: We changed the beginning of the introduction accordingly.\n6. The section on MatK's conserved X domain could use reorganization for better logical\nflow.\nResponse: We adapted the beginning of the paragraph accordingly.\n7. Results line 177 - EMB3120 is annotated at TAIR as - SNOW-WHITE LEAF1, SWL1 (see\nREF of Yue Wang et al Crucial role of SWL1 in chloroplast biogenesis and development in\nArabidopsis thaliana. Plant Cell Rep 43, 135 (2024). https://doi.org/10.1007/s00299-024-\n03210-1.\nResponse: We now cite this reference and the first report of the rice homolog (Hayashi-\nTsugane et al., 2014) at this instance."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the new version of the manuscript, the authors appropriately addressed all of my points.", "reviewer_2": "review to NCOMMS-24-81917A:\nOverall assessment\nIn the revised MS, I find that the authors have responded constructively to the comments. some experimental limitations\nremain obvious (e.g., due to seedling lethality), but these are now more clearly stated, and additional data and analyses\nstrengthen the original work. Major conceptual concerns, including the unsupported model, have been adequately\naddressed. Overall, the revised manuscript is improved and the responses are aligned with the reviewer’s comments. Below\nI attched a point-by-point comments, highlighting where responses are fully aligned, adequately justified despite limitations,\nor still somewhat constrained but still otherwise acceptable.\n• Mixing genetic systems (ABI3 vs amiR lines): Authors acknowledge the confusion, explain biological/technical constraints\n(seedling lethality, tissue limitation), and justify why deep molecular analyses focus on amiR lines compensate by adding\nTEM for amiR lines, expanding proteomics and rRNA analyses, explicitly stating limitations in the Results. Although this part\nmay not fully meet the ideal request (same assays in all lines), the response is scientifically justified which is acceptable in\nmy opinion.\n• Protein-level effects vs RNA defects: Authors provide in their revised MS a quantitative MS/MS proteomics, showing to\nreduced plastid ribosomal proteins, reduced plastome-encoded proteins, and dosage-dependent effects across amiR lines.\nThis is more convincingly addresses the main concern and further strengthens the manuscript.\n• RbcL blot (Fig. 7): I find that the revised MS and authors response adequately addressed the comments. Authors give a\nbiologically coherent explanation, with the timing of induction, differential sensitivity of developing vs mature tissues, and a\nstronger phenotype under early induction. The authors further avoid overinterpretation and acknowledge pleiotropy. This is\nquite convincing and seems consistent with what is known and established in plastid biology.\n• Group II intron background: Authors expanded the introduction as requested.\n• Abrupt transition to MatK discussion: Minor but important stylistic fix implemented as advised.\n• MKIP1 splice variants and locus choice: The revised MS and author response are well aligned with the comments. RNA-\nseq support proteomics evidence, functional complementation, and a clear justification for focusing on AT3G20440.2. I find\nthat this adequately resolves the concern.\n• MKIP1 self-association / paralog ambiguity: Authors clarify peptide mapping ambiguity. They provide additional\nexperimental evidence (tobacco transient IPs) ruling out self-oligomerization. As in the case of the previous comment, this\ndirectly answers my comment.\n• Localization controls: Authors add MitoTracker, DAPI, improved confocal imaging. So trhey fully Address the prior concern.\nThis strengthens the localization claim.\n• Global ribosome biogenesis and translation effects: In my comments I suggested broader evidence (e.g., rRNAs,\ntranslation). Authors add here a comprehensive rRNA northern blots, some proteomics data, comparative logic with known\nribosome mutants, they explicitly frame these as secondary effects, which is scientifically appropriate. While sucrose\ngradients might have been ideal, the combination of data provided herein in the revised MS reasonably “satisfies” the\nconcern.\n• Unsupported model (Fig. 8): Authors fully accept the criticism (of both rev’s) and remove the model. They reason MatK\nbinding with prior literature and clearly separate speculation from data.\n• Minor remarks section: regarding Abstract clarity, Intro verbosity, Terminology, Citation additions, and Rephrasing requests\n- these have been adequately addressed and appropriately revised."}, "author_response": ""}]

Decision letter