Peer review of: Plasma membrane H+-ATPases sustain pollen tube growth and fertilization (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Hoffmann et al. reports genetic and phenotypic characterization of loss of function aha mutants in 
pollen tube growth in Arabidopsis. The authors present pollen tube growth defect of aha6/8/9 
mutants, which correlates with lower cytoplasmic pH values, cytoplasmic pH oscillation profiles 
along the tube, actin organization, and pollen tube shapes. No single mutants of aha6, 7, 8, or 9 
showed phenotypic alterations compared with wildtype plants. On the other hands, they 
demonstrate that triple knockout mutations of aha6/8/9 dramatically reduced pollen tube growth 
thus seed production. Plasma membrane H+-ATPase is an essential ion pump in yeast and plants. 
In the Arabidopsis genome, AHA forms a gene family whereby cell-type specificity as well as 
overall protein expression levels determine the function of each isoforms. In this study, the 
authors examine pollen expressed AHA isoforms. 

In molecular genetic characterization, their results showed that aha6/8/9 triple homozygous 
mutant can be viable. Description and characterization of mutant alleles are confusing. In the 
diagram in Fig 2a, aha8-1 has an insertion at one of the exon with which one would expect this 
allele is null. In Fig. 2b and c, aha8-1 plant expresses AHA8 mRNA and aha8-3 plant appeared to 
be a null mutant. But they have chosen to work aha8-1 which is a weak allele based on their 
characterization. It would be useful if they can provide more explanation for characterization of 
aha8 mutant alleles. They mention quadruple aha6/7/8/9 mutant does not exist. This is an 
ambiguous statement because they did not show numbers of plants screened nor statistics to 
support this. Ideally they should have shown molecular complementation of triple or quadruple 
mutants. Also GFP tagging of AHA6, 8, and 9, shown in Fig. 1a, are they triple mutant 
background? It would make sense if they show AHAx-GFP expression in the mutant background 
and functional complementation. This was not clearly mentioned in the text. Another possibility is 
using yeast heterogenous expression system to test the function of the tagged AHAxs. In Fig. 1b 
and in the text, they describe that the full length AHA6, 8, and 9 do not complement pma1 
knockout yeast strain, because of the presence of the C-terminal auto-inhibitory domain. Is this 
statement true only for AHA6, 8, and 9? Or also applicable to other AHAs? AHA2 or tobacco H+-
ATPase gene expression were reported by the same author and successful complementation was 
shown using pma complementation assay. Rudashevskaya et al. 2012 showed AHA2 full length can 
complement pma1. Please comment on relationship of functional complementation assay in yeast 
and plants. 

Fig.2 f and h. The results shown in seed counts (f) and growth rate (h) do not seem to agree. In 
seed counts, aha6/8 showed the striking defective phenotype, but it does not show defect in the 
growth rate assay. On the other hand, aha6/9 mutant shows the moderate defect in seed counts, 
but reduced growth rate compared with wildtype. Please double check the genotype comparison. If 
this is the case, there seems to be specific roles for AHA8 and AHA9 during pollen tube elongation 
and fertilization, which needs to be discussed. 

For in vitro pollen tube assay, they have used the synthetic media at pH7.5. Does this assay 
reflect what pollen tubes experience in the style in vivo? The original lily pollen paper used pH 5.7. 
It might be useful to mention the evolution of pollen tube assay system, although the space might 
be limited. Can they do their pHluorin assay in the Arabidopsis stigma surface to check pH of the 
apoplast during pollen germination in vivo? 

It would provide a stronger support if they provide data on imaging/measurement of extracellular 
pH, using either pHluorin or pH sensitive reporter dye. As expected, aha mutant shows acidic 
cytoplasmic pH in this report, though cytoplasmic pH can be affected by other transporter activity 
located at endomembranes, which could be difficult to dissect but will be worth to bring readers 
attention. They may also comment that the lack of AHA-GFP signal at the tip. GFP fluorescent 
emission is influenced by pH and other ions such as chloride. Have they tested immuno stain to 

check AHA protein level in addition to imaging methods? 

Three other points severely detract from this reviewers' enthusiasm for the rigor of the work and 
for the rigor of the mechanistic interpretation. 

First, The pump has four products: cytoplasmic pH, cell wall pH (these two form the pH gradient 
but this gradient can be derived from different sets of inside and outside pH values), the pH 
gradient and the membrane potential. As discussed above, although the authors report 
measurements of pH inside no where do i see a report on the membrane potential or the external 
pH.. Thus any interpretation of the role of anion fluxes or cation fluxes or any fluxes without 
knowing how what the changes in the membrane potential and delta pH are as a function of 
genotype and position in the tube and time after germination of tube, are not possible and 
equivocal. 

Second, the measurements of turgor pressure are not really turgor pressure. They are measuring 
the osmotic potential. One needs to directly measure pressure with a pressure probe and then with 
the osmotic potential of the cell contents known, the total water potential can be determined. But 
incipient plasmolysis does not measure turgor pressure. It measures the osmotic potential outside 
the cell at which the cell loses turgor but this outside osmotic potential is not turgor pressure. A 
pressure probe must be used to make any conclusions on turgor pressure. 

Third, and this one may seem trivial but is a bit disturbing since it is the first time this reviewer 
has seen it. The authors refer to AHA's as Autoinhibited H-Atpases..but the term was first coined, 
and has been used consistently since then, to refer to Arabidopsis H+-ATPases...that is why one 
must use NtAHA1 etc to denote the tobacco orthologues, for example (Nt is Nicotiana tabacum). 
While it may be true that many or all of the AHAs have a c terminal domain that acts as an 
autoinibitory domain under some conditions (eg expression in yeast and complementation of yeast 
pma1 knockouts), it is not at all clear to this reviewer that this should be the defining 
characteristic of this set of pumps. What is especially annoying is that this comes out of nowhere 
and for folks following the literature, brings in considerable confusion without a clear explanation 
for why it is put forward. 

In conclusion, while the relationship between tube growth and genotype of the pump seems to be 
striaghtforward the interpretation of this, especially in relation to turgor pressure and anion fluxes, 
without a clear measurement of the turgor pressure at various points in the tube, and without 
measurements of membrane potential, the interpretation and model simply cannot stand. The 
pump's role at the plasma membrane is fundamental for all of the other cotransporters and 
channels that one must be VERY careful about interpreting a phenotype without a precise 
measurement of the properties such as membrane potential and turgor pressure. It is not 
surprising to see cytoskeletal properties altered in the mutant but mechanistically ruling our turgor 
pressure and incorporating anion fluxes specifically wihtout measurements of the real pressure and 
the membrane potential seem to set the rigor of this paper too low the bar of what one expects in 
a paper in Nature Communications. 

Other points: 

- Supplemental figure 1, for mRNA expression profiles and comparisons among AHA6, 7, 8, and 9, 
it will be useful to show the expression levels of the four AHAs in pollen. The current figures shows 
the expression of each genes in various developmental stages, which supports they are pollen 
specific isoforms, but from this figure one cannot directly see a comparison of pollen expression 
among four genes (i.e., which gene has the highest expression in pollen?) 

- Fig.1. The image for AHA9-GFP is truncated at the tip of the pollen tube. It would be nice to be 
able to see the entire pollen tube. 

- Page 7, line 16. Overexpression of AHA7 partially rescued pollen tube defect of aha6/8/9. No 
data shown to support this statement. Also no information provided as to the promoter or plasmid 
construct 

Reviewer #2 (Remarks to the Author): 

The Hoffman et al. study furthers the demonstration of H+-ATPases as key regulator of growth, 
and in this case, the study focuses on polarize growth in pollen tubes, where there is a rich history 
of ionic regulation, in particular by H+ and Ca2+. The study was carried by long standing expert 
groups in H+-ATPases and regulation of ion homeostasis in pollen tubes. 
The premise of the study is straight-forward, based on the characterization of double and triple 
aha mutants in Arabidopsis, ara6 being at the core, its combination with one or two of two 
additional mutatant ahas (8, 9, and to a lesser extent 7). Not surprisingly pollen tube growth 
became increasingly compromised to the extent of reaching close to male sterile (Fig. 1,2) . That’s 
where the physiological aspects of characterizing the H+-related parameters began. While the 
electrophysiology studies were rather extensive, the results were demonstrably presented in 
several summary plots (Fig 3,4). The data trends are generally in agreement with the order of 
combined mutations and the severity of tube growth properties (while not precisely linear, the 
data are reasonably cohesive). As a reviewer far from being an expert in these physiological 
approaches, the summary data presented here is adequate to relay the authors conclusion from 
these data. I’ll leave for the experts to decipher the actual data acquired and the statistically 
analyses. Of Fig. 3,4, I wish Fig. 4C to be more accessible. Supplemental Fig. 9 also. 

Fig. 5 shows disruption in actin accompanies loss of aha-induced pollen tube growth defects. While 
I understand the wish to connect disrupt H+ status to the key structure that support pollen tube 
growth, the demonstration is hardly adequate to rely a direct causal relationship, although it’s hard 
to recommend another target to examine. 

Overall I find it to be a huge amount of meticulously collected data, both on the genetics and 
reproduction characterization and the pollen tube growth and physiology analyses. The findings 
are consistent with what one would expect with AHA deficiency. 

I suggest some efforts in revising sentence structure might help the reading of the manuscript. 
Examples: 

Pg. 2, Lines 11-14: perhaps try :” Tubes lacking AHAt displayed reduced cytosolic pH, tip-to-shank 
protein gradients….. “ the phrase “which were syncrhoized with ossiclations in growth rate”. 
Pg. 4, Lines 9-11: either rearrange the phrases of separate into two sentences. 

Reviewer #3 (Remarks to the Author): 

Involvement of H+ -ATPases in pollen tube growth has long been suggested. However, direct 
evidence linking AHAs with pollen tube growth was lacking due to its high degree of genetic 
redundancy. In this MS, authors are providing solid genetic evidence that PM H+ -ATPases play a 
pivotal role in pollen tube growth. I congratulate authors for taking multiple experimental 
approaches to demonstrate the functional role of AHA6/8/9 in modulating H+ fluxes and 
intracellular pH dynamics. The findings of this study will be highly useful to refine tip-growing 
theories. Overall this MS is well written and all the conclusions are well supported with actual data. 
Though the involvement of AHAs in regulating H+ fluxes is demonstrated in this MS, it would be 
nice to show how these H+ fluxes alter the membrane potential in tip and shank. Based on the H+ 

flux data the tip should show depolarisation and the sank should show hyperpolarisation. The AHA 
mutants’ also should have depolarised membrane potential compare to wild type in shank. 

Some other minor points to consider 
P 5 ln 3, 6 and 7 the figure you are mentioning here is 1a NOT 1b. 
P 7 ln 15-16 Indeed, overexpression of AHA7 partially rescued the pollen tube growth defect of 
aha6/8/9.- No data has been presented to support this claim. 
P21 ln 30-34. It is critical to provide ion-selective cocktail catalogue number used for making H+ 
and Cl– selective electrodes. 
P35 Figure 1 legend. Please modify based on the actual figure. Panel a and b are swapped. 
P 36 Figure 3 legend. On panel C please mention negative sign means influx and the positive sign 
means efflux. 
Please revise supplementary figure 9 legend panel information.