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1. Plant KASH proteins SINE1 and SINE2 have synergistic and antagonistic interactions with actin-branching and actin-bundling factors

   https://doi.org/10.1093/jxb/erad400  

   Moser, Morgan; Groves, Norman_R; Meier, Iris; Murray, ed., James (October 2023, Journal of Experimental Botany)

   Abstract Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of outer nuclear membrane KASH proteins, interacting in the nuclear envelope lumen with inner nuclear membrane SUN proteins and connecting the nucleus and cytoskeleton. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light–dark transitions and abscisic acid (ABA), which requires F-actin reorganization. SINE2 influences actin depolymerization and SINE1 actin repolymerization. The actin-related protein 2/3 (ARP2/3) complex, an actin nucleator, and the plant actin-bundling and -stabilizing factor SCAB1 are involved in stomatal aperture control. Here, we have tested the genetic interaction of SINE1 and SINE2 with SCAB1 and the ARP2/3 complex. We show that SINE1 and the ARP2/3 complex function in the same pathway during ABA-induced stomatal closure, while SINE2 and the ARP2/3 complex play opposing roles. The actin repolymerization defect observed in sine1-1 is partially rescued in scab1-2 sine1-1, while SINE2 is epistatic to SCAB1. In addition, SINE1 and ARP2/3 act synergistically in lateral root development. The absence of SINE2 renders trichome development independent of the ARP2/3 complex. Together, these data reveal complex and differential interactions of the two KASH proteins with the actin-remodeling apparatus and add evidence to the proposed differential role of SINE1 and SINE2 in actin dynamics. 

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2. The Arabidopsis KASH protein SINE3 is involved in male and female gametogenesis

   https://doi.org/10.1007/s00497-024-00508-8  

   Moser, Morgan; Groves, Norman_R; Meier, Iris (September 2024, Plant Reproduction)

   Key messageThe Arabidopsis KASH protein SINE3 is involved in male and female gametophyte development, likely affecting the first post-meiotic mitosis in both cases, and is required for full seed set. AbstractLinker of nucleoskeleton and cytoskeleton (LINC) complexes are protein complexes spanning the inner and outer membranes of the nuclear envelope (NE) and are key players in nuclear movement and positioning. Through their roles in nuclear movement and cytoskeletal reorganization, plant LINC complexes affect processes as diverse as pollen tube rupture and stomatal development and function. KASH proteins are the outer nuclear membrane component of the LINC complex, with conserved C-termini but divergent N-terminal cytoplasmic domains. Of the known Arabidopsis KASH proteins, SUN-INTERACTING NUCLEAR ENVELOPE PROTEIN 3 (SINE3) has not been functionally characterized. Here, we show that SINE3 is expressed at all stages of male and female gametophyte development. It is located at the NE in male and female gametophytes. Loss of SINE3 results in a female-derived seed set defect, withsine3mutant ovules arresting at stage FG1. Pollen viability is also significantly reduced, with microspores arresting prior to pollen mitosis I. In addition, sine3mutants have a minor male meiosis defect, with some tetrads containing more than four spores. Together, these results demonstrate that the KASH protein SINE3 plays a crucial role in male and female gametophyte development, likely affecting the first post-meiotic nuclear division in both cases. 

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3. The Arabidopsis heterotrimeric G‐protein β subunit, AGB 1, is required for guard cell calcium sensing and calcium‐induced calcium release

   https://doi.org/10.1111/tpj.14318  

   Jeon, Byeong Wook; Acharya, Biswa R.; Assmann, Sarah M. (April 2019, The Plant Journal)

   <bold>Summary</bold> Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit,AGB1, is required for four guard cell Ca_oresponses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cytoscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit,GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements ofagb1mutants andagb1/gpa1double‐mutants, as well as those of theagg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast withABA‐regulated stomatal movements, which involveGPA1 andAGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding.AGB1knockouts retained reactive oxygen species andNOproduction, but lostYC3.6‐detected [Ca²⁺]_cytoscillations in response to Ca_o, initiating only a single [Ca²⁺]_cytspike. Experimentally imposed [Ca²⁺]_cytoscillations restored stomatal closure inagb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed thatAGB1 interacts with phospholipase Cs (PLCs), and Ca_oinduced InsP3 production in Col but not inagb1. In sum, G‐protein signaling viaAGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_oapparently require Ca²⁺‐induced Ca²⁺release that is likely dependent on Gβγ interaction withPLCs leading to InsP3 production. 

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4. A role for calcium‐dependent protein kinases in differential CO ₂ ‐ and ABA‐controlled stomatal closing and low CO ₂ ‐induced stomatal opening in Arabidopsis

   https://doi.org/10.1111/nph.17079  

   Schulze, Sebastian; Dubeaux, Guillaume; Ceciliato, Paulo H. O.; Munemasa, Shintaro; Nuhkat, Maris; Yarmolinsky, Dmitry; Aguilar, Jaimee; Diaz, Renee; Azoulay‐Shemer, Tamar; Steinhorst, Leonie; et al (December 2020, New Phytologist)

   Summary Low concentrations of CO₂cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca²⁺and protein phosphorylation in CO₂‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca²⁺into specific phosphorylation events. However, Ca²⁺‐binding proteins that function in the stomatal CO₂response remain unknown.Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed.We isolatedcpkquintuple mutants and analyzed stomatal movements in response to CO₂, light and abscisic acid (ABA). Interestingly, we found thatcpk3/5/6/11/23quintuple mutant plants, but not other analyzedcpkquadruple/quintuple mutants, were defective in high CO₂‐induced stomatal closure and, unexpectedly, also in low CO₂‐induced stomatal opening. Furthermore, K⁺‐uptake‐channel activities were reduced incpk3/5/6/11/23quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO₂‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targetedcbl1/4/5/8/9quintuple mutant plants.Our findings describe combinatorialcpkmutants that function in CO₂control of stomatal movements and support the results of classical studies showing a role for Ca²⁺in this response. 

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5. Abscisic acid driven stomatal closure during drought in anisohydric Fagus sylvatica

   Kane, Cade; McAdam, Scott (September 2023, The Journal of plant hydraulics)

   Stomatal closure limits transpiration during drought, restricting water potential decline and delaying the onset of embolism. While critical for ensuring survival during drought, the mechanisms driving stomatal closure during drought remain equivocal. The hormone abscisic acid (ABA) will close stomata in seed plants and is synthesized as leaf turgor declines. ABA driven stomatal closure during drought is particularly apparent in species that are more isohydric. In contrast, in species that have a more anisohydric response to drought, like Fagus sylvatica, the importance of ABA in driving stomatal closure during drought is often overlooked or excluded, in place of a hypothesized passive, water potential driven stomatal closure. Here we investigated whether ABA drives stomata closure during a mid-summer drought in field grown F. sylvatica. We found that as leaf water potential declines during a drought, foliage abscisic acid (ABA) levels increase considerably and stomata close. ABA levels in leaves increase as water potentials decline to within 0.3 MPa of turgor loss point, when stomata close. Foliage ABA levels correlate with stomatal conductance throughout a drought and post-drought period. From these results we argue that it is hard to exclude increased ABA levels driving stomatal closure during drought in the anisohydric species F. sylvatica. 

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